Methods of expanding embryonic stem cells in a suspension culture

ABSTRACT

A method of expanding and maintaining human embryonic stem cells (ESCs) in an undifferentiated state by culturing the ESCs in a suspension culture under culturing conditions devoid of substrate adherence is provided. Also provided are a method of deriving ESC lines in the suspension culture and methods of generating lineage-specific cells from ESCs which were expanded in the suspension culture of the present invention.

RELATED APPLICATIONS

This Application is a division of U.S. patent application Ser. No.14/720,853 filed on May 25, 2015, which is a continuation of U.S. patentapplication Ser. No. 12/309,817 filed on Aug. 14, 2009, which is aNational Phase of PCT Patent Application No. PCT/IL2007/000970 havingInternational Filing Date of Aug. 2, 2007, which claims the benefit ofU.S. Provisional Patent Application Nos. 60/840,692 filed on Aug. 29,2006 and 60/834,795 filed on Aug. 2, 2006. The contents of the aboveapplications are all incorporated herein by reference.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 71791SequenceListing.txt, created on Nov. 15,2017, comprising 11,089 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a method of expanding and maintainingembryonic stem cells (ESCs) in an undifferentiated state in a suspensionculture, and more particularly, to methods of using such ESCs for thegeneration of lineage-specific cells which can be used for cell-basedtherapy.

Human embryonic stem cells (hESCs) are proliferative, undifferentiatedstem cells capable of differentiating into cells of all three embryonicgerm layers. As such, hESCs hold promise for various applicationsincluding cell-based therapy, pharmaceutical screening, identificationof drug targets and cell-based compound delivery which require almostindefinite amounts of proliferating, yet pluripotent hESCs.

To facilitate the exploitation of hESCs in both cell-based therapy anduse in the pharmaceutical industry for drug screening, identification ofdrug targets and cell-based compound delivery, hESCs cultures should bescaled-up and optimized. However, culturing of hESCs on any of thecurrently available 2-dimensional (2-D) culturing systems (i.e., feederlayers or feeder-free matrices) limits the expansion capacity of thecells. On the other hand, when ESCs are removed from their feeder layersor feeder-free matrices and transferred to common suspension cultures,the cells loose their undifferentiated state and rapidly differentiate(Thomson et al., 1998). Thus, culturing of hESCs in suspension in Petridishes usually results in the formation of aggregates containingdifferentiating cells termed embryoid bodies (EBs) [Itskovitz-Eldor etal, 2000].

To overcome such limitations, Fok and Zandstra (Fok E Y, and Zandstra PW, Stem Cells. 2005, 23: 1333-42) developed stirred-suspension culturesin which the ESCs are attached to glass microcarriers. However, althoughESCs cultured under such conditions exhibited typical ESC expressionpatterns and retained the developmental potential of the starting cellpopulation, the technical difficulties associated with adherence anddissociation of the ESCs from the microcarrier surface limit therobustness potential of such a culturing method. Another study byGerecht-Nir and Itskovitz-Eldor (disclosed in PCT/IL03/01017) describesa dynamic culturing system for differentiating embryoid bodies orexpanding ESCs under non-differentiation conditions. In this system,ESCs are seeded in a bioreactor designed to exert random gravity forces.However, PCT/IL03/01017 does not teach non-dynamic suspension culturesystems. Another study by Cormier J. et al. (Tissue engineering 12:3233-3245, 2006) describes culturing for 6 days of mouse embryonic stemcells (mESCs) in a suspension culture in the presence of leukemiainhibitory factor (LIF) and bovine serum under constant agitation. In alater publication (Zur Nieden N I, et al., 2007; J. of Biotechnology129: 421-432) the authors reported that mESCs cultured in suspensionunder static conditions and using trypsin for passaging every 2 daysexhibited a sharp decrease in the expression of undifferentiated markerssuch as Oct-4 and failed to maintain pluripotency as detected by theexpression of early ectodermal and endodermal differentiation markers.In addition, the doubling time of the mESCs that were cultured in thedynamic or static suspension cultures was only 15 hours (Zur Nieden., etal., Supra), which may lead to chromosomal instability and abnormality(Cowan C A., et al., 2004, N. Engl. J. Med. 350: 1353-1356). Inaddition, in contrast to mESCs, it is known that LIF cannot maintainhuman ESCs in an undifferentiated state (Thomson et al, 1998; Reubinofet al, 2000). Thus, to date, continuous culturing of undifferentiatedhuman ESCs in suspension under conditions devoid of substrate adherence(e.g., a carrier) was never demonstrated.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, a method of obtaining a scalable culture of hESCsdevoid of the above limitations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of expanding and maintaining human embryonic stem cells in anundifferentiated state, the method comprising culturing the embryonicstem cells in a suspension culture under culturing conditions devoid ofsubstrate adherence and which allow expansion of the embryonic stemcells in the undifferentiated state, thereby expanding and maintainingthe embryonic stem cells in the undifferentiated state.

According to another aspect of the present invention there is provided amethod of deriving an embryonic stem cell line, the method comprising:(a) obtaining an embryonic stem cell from a pre-implantation stageblastocyst, post-implantation stage blastocyst and/or a genital tissueof a fetus; and (b) culturing the embryonic stem cell in a suspensionculture, under culturing conditions which allow expansion of theembryonic stem cells in an undifferentiated state; thereby deriving theembryonic stem cell line.

According to yet another aspect of the present invention there isprovided a method of generating lineage-specific cells from humanembryonic stem cells, the method comprising: (a) culturing the humanembryonic stem cells in a suspension culture under culturing conditionswhich allow expansion of the human embryonic stem cells in anundifferentiated state to thereby obtain expanded, undifferentiatedhuman embryonic stem cells; and (b) subjecting the expanded,undifferentiated human embryonic stem cells to culturing conditionssuitable for differentiating and/or expanding lineage specific cells;thereby generating the lineage-specific cells from the human embryonicstem cells.

According to still another aspect of the present invention there isprovided a method of generating embryoid bodies from human embryonicstem cells, the method comprising: (a) culturing the human embryonicstem cells in a suspension culture under culturing conditions whichallow expansion of the human embryonic stem cells in an undifferentiatedstate to thereby obtain expanded, undifferentiated human embryonic stemcells; and (b) subjecting the expanded, undifferentiated human embryonicstem cells to culturing conditions suitable for differentiating thehuman embryonic stem cells to embryoid bodies; thereby generating theembryoid bodies from the human embryonic stem cells.

According to an additional aspect of the present invention there isprovided a method of generating lineage-specific cells from humanembryonic stem cells, the method comprising: (a) culturing the humanembryonic stem cells in a suspension culture under culturing conditionswhich allow expansion of the human embryonic stem cells in anundifferentiated state to thereby obtain expanded, undifferentiatedhuman embryonic stem cells; (b) subjecting the expanded,undifferentiated human embryonic stem cells to culturing conditionssuitable for differentiating the expanded, undifferentiated humanembryonic stem cells to embryoid bodies; and (c) subjecting cells of theembryoid bodies to culturing conditions suitable for differentiatingand/or expanding lineage specific cells; thereby generating thelineage-specific cells from the human embryonic stem cells.

According to yet an additional aspect of the present invention there isprovided a culture medium comprising a soluble interleukin-6 receptor(sIL6R) and soluble interleukin-6 (IL6), wherein the sIL6R is present ata concentration of at least 10 nanogram per milliliter (ng/ml).

According to still an additional aspect of the present invention thereis provided a culture medium comprising at least 2000 units permilliliter (u/ml) leukemia inhibitor factor (LIF).

According to a further aspect of the present invention there is provideda cell culture comprising cells and the culture medium of the presentinvention.

According to yet a further aspect of the present invention there isprovided a cell culture comprising human embryonic stem cells and aculture medium which comprises at least 1000 u/ml of leukemia inhibitorfactor (LIF).

According to further features in preferred embodiments of the inventiondescribed below, the culture medium is capable of maintaining the humanembryonic stem cells in an undifferentiated state.

According to still further features in the described preferredembodiments the cells are embryonic stem cells.

According to still further features in the described preferredembodiments the embryonic stem cell is a human embryonic stem cell.

According to still further features in the described preferredembodiments the embryonic stem cell is a primate embryonic stem cell.

According to still further features in the described preferredembodiments the culture medium is capable of maintaining the embryonicstem cells in an undifferentiated state.

According to still further features in the described preferredembodiments, the expansion comprises obtaining at least 9×10¹⁵ cellsfrom a single embryonic stem cell following 3 months.

According to still further features in the described preferredembodiments culturing is effected under conditions devoid of substrateadherence.

According to still further features in the described preferredembodiments the suspension culture is serum-free, serumreplacement-free, xeno-free, feeder-free and protein carrier-free.

According to still further features in the described preferredembodiments the medium comprises a TGFβ isoform.

According to still further features in the described preferredembodiments a medium of the suspension culture comprises an IL6RIL6chimera.

According to still further features in the described preferredembodiments culturing is effected under xeno-free conditions.

According to still further features in the described preferredembodiments the TGFβ isoform is a TGFβ isoform 1 (TGFβ₁).

According to still further features in the described preferredembodiments the TGFβ isoform is a TGFβ isoform 3 (TGFβ₃).

According to still further features in the described preferredembodiments the TGFβ₁ is present at a concentration of at least 0.06ng/ml.

According to still further features in the described preferredembodiments the TGFβ₁ is present at a concentration of 0.12 ng/ml.

According to still further features in the described preferredembodiments the TGFβ₃ is present at a concentration of at least 0.5ng/ml.

According to still further features in the described preferredembodiments the TGFβ₃ is present at a concentration of 2 ng/ml.

According to still further features in the described preferredembodiments the medium further comprises basic fibroblast growth factor(bFGF).

According to still further features in the described preferredembodiments the bFGF is present at a concentration of at least 2 ng/ml.

According to still further features in the described preferredembodiments the bFGF is present at a concentration of at least 4 ng/ml.

According to still further features in the described preferredembodiments the IL6RIL6 chimera is present at a concentration of atleast 25 ng/ml.

According to still further features in the described preferredembodiments the sIL6R is present at a concentration of 15-30 ng/ml.

According to still further features in the described preferredembodiments the medium comprises a soluble interleukin-6 receptor(sIL6R), wherein the sIL6R is present at a concentration of 15-30 ng/ml.

According to still further features in the described preferredembodiments the medium further comprises soluble interleukin-6 (IL6).

According to still further features in the described preferredembodiments the medium comprises leukemia inhibitor factor (LIF),wherein the LIF is present at a concentration of at least 2000 units permilliliter (u/ml).

According to still further features in the described preferredembodiments the medium comprises leukemia inhibitor factor (LIF),wherein the LIF is present at a concentration of at least 1000 units permilliliter (u/ml).

According to still further features in the described preferredembodiments the medium further comprises serum or serum replacement.

According to still further features in the described preferredembodiments the serum or serum replacement is present at a concentrationof at least 10%.

According to still further features in the described preferredembodiments the method further comprising isolating lineage specificcells following step (b).

According to still further features in the described preferredembodiments isolating lineage specific cells is effected by a mechanicalseparation of cells, tissues and/or tissue-like structures containedwithin the embryoid bodies.

According to still further features in the described preferredembodiments isolating lineage specific cells is effected by subjectingthe embryoid bodies to differentiation factors to thereby inducedifferentiation of the embryoid bodies into lineage specificdifferentiated cells.

According to still further features in the described preferredembodiments the protein carrier is albumin.

According to still further features in the described preferredembodiments the embryonic stem cells cultured in the suspension cultureexhibit normal chromosomal karyotype following at least 2 passages.

According to still further features in the described preferredembodiments the embryonic stem cells cultured in the suspension cultureexhibit normal chromosomal karyotype following at least 14 passages.

According to still further features in the described preferredembodiments the embryonic stem cells cultured in the suspension cultureexhibit a doubling time of at least 20 hours.

According to still further features in the described preferredembodiments the undifferentiated state is maintained for at least 5passages in culture.

According to still further features in the described preferredembodiments the undifferentiated state is maintained for at least 15passages in culture.

According to still further features in the described preferredembodiments the culturing conditions are non-dynamic culturingconditions.

According to still further features in the described preferredembodiments maintaining the embryonic stem cells in an undifferentiatedstate is effected in a suspension culture.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a method of expanding andmaintaining embryonic stem cells in an undifferentiated state in asuspension culture.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1a-1d are photomicrographs depicting the morphology ofundifferentiated hES colonies and hES single cells grown in variousculture systems in the presence of the TGFβ-containing culture media.FIG. 1a —I4 hESCs cultured for 28 passages on a Matrigel™ matrix in thepresence of the D1 medium; FIG. 1b —I4 hESCs cultured for 9 passages onMEFs in the presence of the HA16 medium; FIG. 1c —I4 hESCs cultured for20 passages on foreskins fibroblasts in the presence of the D2 medium;FIG. 1d —I4 hESCs cultured for 11 passages on a human fibronectin matrixin the presence of the D2 medium. Note the undifferentiated morphologyafter prolonged culturing with the unique serum-free, serumreplacement-free and protein carrier-free TGFβ-containing media types.Magnifications were ×15 for FIGS. 1a -1 d.

FIGS. 2a-2c are photomicrographs depicting undifferentiated hES coloniesstained with surface markers specific to the hESC undifferentiatedstage. I4 hESCs were cultured for 36 passages on a Matrigel™ matrix inthe presence of the D1 medium and stained with TRA-1-60 (FIG. 2a ),SSEA4 (FIG. 2b ) and TRA-1-81 (FIG. 2c ); Magnifications were ×20 forFIGS. 2a -2 c.

FIGS. 3a-3b are photomicrographs depicting the derivation of a new hESCline under xeno-free conditions on foreskin fibroblasts using the HA16medium. FIG. 3a —the cultured embryo at first passage (p-1), arrowpoints at the inner cell mass (ICM); FIG. 3b —the isolated ICM atpassage 2 (p-2). Magnifications were ×20 for FIGS. 3a -3 b.

FIGS. 4a-4c are photomicrograph depicting immunostaining of hESCscultured for three passages in suspension in the presence of the D2medium. Shown is the expression of Oct4 (FIG. 4a ), TRA-1-60 (FIG. 4b )and TRA-1-81 (FIG. 4c ); Magnifications were ×63 for FIGS. 4a -4 c.

FIGS. 5a-5g are photomicrographs depicting histological sections andmorphology of suspended hESCs culture. FIG. 5a —Histology of a hESCclump (the I4 hESC line) cultured for 3 passages in suspension in thepresence of the D1 medium and stained with H&E. Note that the hESC clumpis homogeneous, containing small cells with large nuclei typical forhESCs morphology. Magnification was ×20. FIGS. 5b-5c —I4 hESCs werecultured for 3 passages in suspension in the presence of the D2 mediumand were then re-cultured on MEFs. Shown is the morphology of thecolonies (as photographed using an inverted microscope) afterre-culturing on MEFs (magnification ×15 for FIGS. 15b-15c ). Note thetypical undifferentiated morphology of the hESCs. FIGS. 5d-15e —I4 hESCswere cultured for 16 passages in suspension in the presence of theCM100F medium and were then re-cultured on MEFs. Shown is the morphologyof colonies after re-culturing on MEFs. Note the typicalundifferentiated morphology of the hESCs. Magnification ×15 for FIGS.15d-15e . FIGS. 5f-g —I4 hESCs were cultured for 7 passages insuspension in the presence of the HA19 medium (FIG. 5f ) or for 10passages in the presence of the CM100F medium (FIG. 5g ). Magnification×10 for FIGS. 15f -15 g.

FIGS. 6a-6d are RT-PCR analyses depicting the expression ofrepresentative genes of the undifferentiated state of hESCs cultured insuspension in the presence of the HACM100, CM100F or the HA19 medium.Lane 1—I-4 hESCs cultured for 1 passage in suspension in the presence ofthe HACM100 medium (serum or serum replacement-free, IL6RIL6-containingmedium). Lane 2—I-4 hESCs cultured for 1 passage in suspension in thepresence of the CM100F medium (IL6RIL6 and serum replacement-containingmedium). Lane 3—I-4 hESCs cultured for 7 passages in suspension in thepresence of the HA19 medium (serum or serum replacement-free, proteincarrier-free, TGFβ₃-containing medium). Lane 4—I-4 hESCs cultured for 2passages in suspension in the presence of the HA19 medium and thenre-cultured on MEFs for additional 6 passages. FIG. 6a —Oct4; FIG. 6b—Rex1; FIG. 6c —Sox2; FIG. 6d —Nanog; RT mix were tested and foundnegative for all tested genes. All samples were tested for β-actin andwere found evenly positive.

FIGS. 7a-7f are photomicrographs depicting the morphology of hESCscultured in suspension under non-dynamic conditions (i.e., staticculture) or 2-dimensional (2D) cultures in the presence of the CM100Fmedium (including the IL6RIL6 chimera) (unless stated otherwise). FIG.7a —Phase contrast image of an undifferentiated hESC colony from I3 cellline cultured for 12 passages on human fibronectin (a 2-D culture). Bar200 μM; FIG. 7b —Image of neurosphere-like structures representing adifferentiation “background” occurring in up to 5% of I6 hES cells whencultured in suspension. Bar 300 μM; FIG. 7c —Undifferentiated I3 hEScells, cultured in suspension for 43 passages. Bar 300 μM; FIG. 7d-Histological section of a clump of undifferentiated cells from I3 hEScells cultured in suspension for 32 passages. Bar 50 μM; FIG. 7e —Phasecontrast image of a hESC colony formed by I3 cells cultured for 10passages in suspension and re-cultured on MEFs (passage one with MEFs).Bar 200 μM; FIG. 7f —A hESC colony formed by I3 cells cultured for 36passages in suspension and re-cultured on fibronectin (passage 10). Bar150 μM.

FIGS. 8a-8d are fluorescent immunostaining analyses depicting theexpression of undifferentiated markers by hESCs cultured in suspensionunder non-dynamic conditions in the presence of the CM100F medium(including the IL6RIL6 chimera). FIG. 8a —hESC line I3 cultured for 42passages in suspension and stained with anti-Oct4 antibodies. Bar 200μM; FIG. 8b —hESC line I3 cultured for 42 passages in suspension andstained with anti-TRA-1-60 antibodies. 200 μM; FIG. 8c —hESC line I3cultured for 32 passages in suspension and stained with anti-TRA-1-81antibodies. 150 μM; FIG. 8d —hESC line I3 cultured for 32 passages insuspension and stained with anti-SSEA4 antibodies. 200 μM.

FIG. 9 depicts RT-PCR analyses demonstrating the expression ofundifferentiated markers (Oct4, Nanog, Rex1, FGF4 and Sox2) in hESCscultured in suspension under non-dynamic conditions in the presence ofthe CM100F medium. The I4 hESCs were cultured for 10 (10 p), 15 (15 p)and 20 (20 p) passages in suspension. Similar results were demonstratedfor I3 and I6 hESCs when cultured in suspension in the same culturemedium, each for 10, 15 and 20 passages (data not shown). RT mix for allgenes were negative.

FIGS. 10a-10c are flow cytometry analyses of hESCs cultured insuspension under non-dynamic conditions in the presence of the CM100Fmedium and stained with SSEA4. FIG. 10a —the I6 at passage 20 insuspension; FIG. 10b —the I4 at passage 30 in suspension; FIG. 10c —theI3 at passages 34 in suspension. The percentages of SSEA4-positive cells(indicating undifferentiated cells) in each cell culture were asfollows: I6, 94.7%; I4, 94.5%; I3 87.8%. It should be noted that theclumps of differentiated hESCs in the I3 culture (which consisted of 12%of the cells at passage 34) were removed from the culture and followingadditional 3 passages 95% of the I3 hESCs expressed the SSEA4 marker(data not shown). These results demonstrate that as in 2-D cultures itis possible to remove differentiated colonies and continue culturing ofundifferentiated human ESCs.

FIGS. 11a-11b are real time PCR analyses depicting relative expressionof Oct4 in I6 (FIG. 11a ) and I4 (FIG. 11b ) hESCs that were culturedfor 10 passages in suspension under non-dynamic conditions in thepresence of the CM100F medium. The expression levels were compared tocells from the same cell line cultured continuously on MEFs, which wasused as calibrator. Similar results were obtained when cells culturedfor 15 and 20 passages in suspension were used (data not shown).

FIGS. 12a-12d are photomicrographs depicting representative histologicalsections of EBs (FIG. 12a ) and teratomas (FIGS. 12b-d ). FIG. 12a—14-days-old cystic EB formed by I4 hESCs cultured for 8 passages insuspension under non-dynamic conditions in the presence of the CM100Fmedium. Bar 200 μM. Teratomas sections formed by I4 hESCs cultured for 9passages in suspension in the presence of the CM100F medium createdtissues representing of the three embryonic germ layers, including;myelinated nerve (ectoderm) (FIG. 12b ), cartilage tissue (mesoderm)(FIG. 12c ), and secretory glands—like structures (endoderm) (FIG. 12d). Bar 250 μM for FIG. 12a , and 200 μM for FIGS. 12b -12 d.

FIGS. 13a-13h depict cell growth (FIGS. 13a-d ) and apoptosis (FIGS.13e-h ). I4 hESCs cultured for more than 20 passages in suspension undernon-dynamic conditions in the presence of the CM100F medium were used tomeasure the culture system kinetics. The cells were cultured withoutsplitting for 14 days and the following parameters were measured:increase in clumps diameter (measured in μm) during 14 days ofcontinuous culture (FIG. 13a ); clumps cultured for 2, 6 and 14 daysrepresenting the increase in size (FIGS. 13b-13d ). Bar 300 μM;apoptosis percentage of cells cultured for 14 days in suspension (FIG.13e ); and apoptotic cells within clumps cultured for 2, 6 and 14 days(FIGS. 13f-13h ). Note that apoptotic cells in 14 days old clumps areconcentrated at the center. Bar 150 μM.

FIGS. 14a-14h are photomicrographs depicting dynamic culture usingErlenmeyer's. FIGS. 14a —I3 hESC clumps cultured for 1 month inErlenmeyer in the presence of the CM100F medium. Bar 400 μM; FIG. 14b—colony formed by the cells of FIG. 14a after re-culturing for 1 passage(about 5 days) on MEFs. Bar 200 μM. FIGS. 14c-14e —Images of fluorescentimmunostaining analyses of I3 hESCs cultured for 4 months in Erlenmeyerin the presence of the CM100F medium using Oct4 (FIG. 14c ), SSEA4 (FIG.14d ), and TRA-1-60 (FIG. 14e ). Note that the cultured hESCs werepositively stained with Oct4, SSEA4 and TRA-1-60, markers of theundifferentiated state. Size bars 200 μM. When the cells (after 1 monthof culture in Erlenmeyer) were transferred to serum containing mediumthey formed EBs. EB were re-plated on Gelatin and positively stainedwith β-tubulin (FIG. 140, troponin (FIG. 14g ), and PSA-NCAM (FIG. 14h). Size bars 100 μM.

FIGS. 15a-15d are Western blot analyses for STAT3 (FIG. 15b ),phosphorylated STAT3 (FIG. 15a ), gp130 (FIG. 15c ) and β-actin(control) (FIG. 15d ), depicting possible involvement of the IL6RIL6chimera in cells self-maintenance while cultured in suspension undernon-dynamic conditions in the presence of the CM100F medium. Human ESCSwere cultured for 24 hours in the CM100F medium without the IL6RIL6chimera and then in CM100F with the chimera as indicated for 0, 30minutes, 180 minutes and 24 hours. Trigger experiment demonstratedincrease in proteins expression 30 minutes after retrieving the IL6RIL6chimera, which holds after 24 hours. Lane 1—I3 cultured for 44 passagesin suspension in the presence of the IL6RIL6 chimera; lane 2—I3 cellscultured 37 passages in suspension 24 hours after removing the chimera;lane 3—I3 cells cultured 37 passages in suspension 30 minutes after thechimera was returned to the medium; lane 4-3 hours after the chimera wasreturned to the medium and lane 5-24 hours after the chimera wasreturned to the culture medium.

FIG. 16 is a graph depicting the percentages of differentiating clumpswhile culturing in suspension under non-dynamic conditions in thepresence of the CM100F medium with increasing concentrations ofanti-gp130 added to culture medium. Note the increase in celldifferentiation following increasing concentrations of the anti-gp 130antibody.

FIGS. 17a-17b are images of clumps depicting hESC clump morphology whileculturing in the presence of the CM100F medium and 250 ng/ml of theanti-gp 130 antibody. FIG. 17a —depicts morphology of undifferentiatedhESC clumps in the presence of the antibody. FIG. 17b —depictsmorphology of differentiated hESC clump in the presence of the antibody.Bar 150 μM.

FIGS. 18a-18e depict undifferentiated human ESCs cultured in suspension(under non-dynamic conditions with the yFIL25+ (FIG. 18a ), yFL1 (FIG.18b ), TLF (FIGS. 18c and e ) and yFL3 (FIG. 18d ) culture media. FIG.18a —Clumps of I4 cells cultured for 16 passages in suspension withyFIL25+ medium; FIG. 18b -Clumps of I3 cells cultured for 18 passages insuspension with yFL1 medium; FIG. 18c —hESCs colony from I3 culturedwith TLF on MEFs for 13 passages after 10 passages in suspension; FIG.18d —Clumps of I3 cells cultured for 1 passage in suspension with yFL3medium; FIG. 18e —Clumps of I4 cells cultured for 18 passages insuspension with TLF medium.

FIGS. 19a-19d are photomicrographs depicting undifferentiated human ESCscultured under non-dynamic conditions (static) in suspension in thepresence of the yFIL25+ (FIG. 19a ), TLF (FIGS. 19b-19c ) and yFL3 (FIG.19d ). FIG. 19a —is a photomicrograph depicting clumps of I4 hESCscultured for 18 passages in suspension with yFIL25+ medium and stainedwith Oct4; FIG. 19b —is a photomicrograph depicting clumps of I4 humanESCs cultured for 31 passages in suspension with TLF medium and stainedwith SSEA4; FIG. 19c —is a photomicrograph depicting clumps of I4 cellscultured for 31 passages in suspension with TLF medium and stained withTRA-1-60; FIG. 19d —is a photomicrograph depicting clumps of I4 cellscultured for 18 passages in suspension with yFL3 medium and stained withTRA-1-81.

FIG. 20 is a photomicrograph depicting EBs formed from I4 human ESCswhich were cultured for 24 passages in suspension under staticconditions with TLF medium. For EB formation, the hESCs were transferredto serum containing medium (which is devoid of the LIF and TGFβ1).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention is of a method of expanding and maintainingembryonic stem cells (ESCs) in the undifferentiated state in asuspension culture. In addition, the present invention is of methods ofgenerating lineage-specific cells from ESCs which were expanded by themethod of the present invention and which can be used cell-basedtherapy.

The principles and operation of the method of expanding and maintainingESCs in a suspension culture according to the present invention may bebetter understood with reference to the drawings and accompanyingdescriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

To facilitate the exploitation of hESCs in both cell-based therapy anduse in the pharmaceutical industry for drug screening, identification ofdrug targets and cell-based compound delivery, hESCs cultures should bescaled-up and optimized. However, culturing of hESCs on any of thecurrently available 2-dimensional (2-D) culturing systems (i.e., feederlayers or feeder-free matrices) limits the expansion capacity of thecells. On the other hand, when ESCs are removed from their feeder layersor feeder-free matrices and transferred to common suspension cultures,the cells loose their undifferentiated state and rapidly differentiate(Thomson et al., 1998).

To overcome such limitations, Fok and Zandstra (Fok E Y, and Zandstra PW, Stem Cells. 2005, 23: 1333-42) developed stirred-suspension culturesin which the ESCs are attached to glass microcarriers. However, althoughESCs cultured under such conditions exhibited typical ESC expressionpatterns and retained the developmental potential of the starting cellpopulation, the technical difficulties associated with adherence anddissociation of the ESCs from the microcarrier surface limits therobustness potential of such a culturing method. Another study byGerecht-Nir and Itskovitz-Eldor (disclosed in PCT/IL03/01017) describesa dynamic culturing system for differentiating embryoid bodies orexpanding ESCs under non-differentiation conditions. In this system ESCsare seeded in a bioreactor designed to exert random gravity forces.However, PCT/IL03/01017 does not teach non-dynamic suspension culturesystems. Another study by Cormier J. et al. (Tissue engineering 12:3233-3245, 2006) describes culturing for 6 days of mouse embryonic stemcells (mESCs) in a suspension culture in the presence of leukemiainhibitory factor (LIF) and bovine serum under constant agitation. In alater publication (Zur Nieden N I, et al., 2007; J. of Biotechnology129: 421-432) the authors reported that mESCs cultured in suspensionunder static conditions lost their undifferentiated and pluripotentstate. In addition, the doubling time of the mESCs that were cultured inthe dynamic or static suspension cultures using trypsin for passagingevery 2 days was only 15 hours (Zur Nieden., et al., Supra), which maylead to chromosomal instability and abnormality (Cowan C A., et al.,2004, N. Engl. J. Med. 350: 1353-1356). In addition, in contrast tomESCs, it is known that LIF cannot maintain human ESCs in anundifferentiated state (Thomson et al, 1998; Reubinof et al, 2000).Thus, to date, continuous culturing of undifferentiated human ESCs insuspension under conditions devoid of substrate adherence (e.g., acarrier) was never demonstrated.

While reducing the present invention to practice, the present inventorshave uncovered, through laborious experimentations, that hESCs can becultured in the undifferentiated state in a suspension culture devoid ofsubstrate adherence and that cells cultured in such conditions maintainall typical hESC characteristics including unlimited proliferation inthe undifferentiated state while preserving the pluripotent capacity.

As is shown in FIGS. 4a-4c, 5a-5g, 6a-6d, 8a-8d , 9, 10 a-10 c, 11 a-11b and 18 a-18 e and described in Examples 2, 3 and 4 of the Examplessection which follows, hESCs cultured in a suspension culture devoid ofsubstrate adherence in the presence of a TGF-beta [β]-containing media(e.g., the D1, D2 or HA19 medium), the IL6RIL6 chimera-containing medium(e.g., CM100F or HACM100), soluble IL6 receptor and IL6 (e.g., theyFIL25 medium), or leukemia inhibitory factor (LIF) (e.g., the yFL1,yFL2 or yFL3 media) exhibited typical hESC morphology (e.g., round cellswith large nuclei; for example FIGS. 5a-g, 18a-e ) and expressedhESCs-specific markers of the undifferentiated state such as Oct 4,TRA-1-60, TRA-1-81, SSEA4, Rex1, Sox2, Nanog and FGF4 (FIGS. 4a-4c,6a-6d, 8a-8d , 9, 10 a-10 c, 11 a-11 b and data not shown). In addition,hESCs cultured in the suspension cultures maintained their pluripotentcapacity as evidenced by their ability to form embryoid bodies (EBs) orteratomas containing representative tissues of all three embryonic germlayers (FIGS. 12a-d and data not shown). Thus, these resultsdemonstrate, for the first time, a method of obtaining a scalableculture of hESCs in a defined, xeno-free medium which is suitable forcell-based therapy.

Thus, according to one aspect of the present invention there is provideda method of expanding and maintaining embryonic stem cells in anundifferentiated state. The method is effected by culturing theembryonic stem cells in a suspension culture under culturing conditionsdevoid of substrate adherence and which allow expansion of the embryonicstem cells in the undifferentiated state, thereby expanding andmaintaining the embryonic stem cells in the undifferentiated state.

As used herein the phrase “embryonic stem cells” refers to embryoniccells which are capable of differentiating into cells of all threeembryonic germ layers (i.e., endoderm, ectoderm and mesoderm), orremaining in an undifferentiated state. The phrase “embryonic stemcells” may comprise stem cells obtained from the embryonic tissue formedafter gestation (e.g., blastocyst) before implantation (i.e., apre-implantation blastocyst), extended blastocyst cells (EBCs) which areobtained from a post-implantation/pre-gastrulation stage blastocyst, andembryonic germ (EG) cells which are obtained from the genital tissue ofa fetus any time during gestation, preferably before 10 weeks ofgestation. Preferred embryonic stem cells according to this aspect ofthe present invention are of a human or primate (e.g., monkey) origin.The embryonic stem cells of the present invention can be obtained usingwell-known cell-culture methods. For example, human embryonic stem cellscan be isolated from human blastocysts. Human blastocysts are typicallyobtained from human in vivo preimplantation embryos or from in vitrofertilized (IVF) embryos. Alternatively, a single cell human embryo canbe expanded to the blastocyst stage. For the isolation of human ES cellsthe zona pellucida is removed from a 5-7 day-old blastocyst and theinner cell mass (ICM) is isolated by immunosurgery, in which thetrophectoderm cells are lysed and removed from the intact ICM by gentlepipetting. The ICM is then plated in a tissue culture flask containingthe appropriate medium which enables its outgrowth. Following 9 to 15days, the ICM derived outgrowth is dissociated into clumps either by amechanical dissociation or by an enzymatic degradation and the cells arethen re-plated on a fresh tissue culture medium. Colonies demonstratingundifferentiated morphology are individually selected by micropipette,mechanically dissociated into clumps, and re-plated. Resulting ES cellsare then routinely split every 4-7 days. For further details on methodsof preparing human ES cells see Thomson et al., (U.S. Pat. No.5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998;Proc. Natl. Acad. Sci. USA 92: 7844, 1995); Bongso et al., (Hum Reprod4: 706, 1989); and Gardner et al., (Fertil. Steril. 69: 84, 1998).

It will be appreciated that commercially available embryonic stem cellscan also be used with this aspect of the present invention. Human ESCscan be purchased from the NIH human embryonic stem cells registry(http://escr.nih.gov). Non-limiting examples of commercially availableembryonic stem cell lines are BG01, BG02, BG03, BG04, CY12, CY30, CY92,CY10, TE03 and TE32.

Extended blastocyst cells (EBCs) can be obtained from a blastocyst of atleast nine days post fertilization at a stage prior to gastrulation.Prior to culturing the blastocyst, the zona pellucida is digested [forexample by Tyrode's acidic solution (Sigma Aldrich, St Louis, Mo., USA)]so as to expose the inner cell mass. The blastocysts are then culturedin vitro as whole embryos for at least nine and no more than fourteendays post fertilization (i.e., prior to the gastrulation event) usingstandard embryonic stem cell culturing methods. For further details ofmethods of obtaining EBCs see WO2006/040763 to the present inventors.

EG cells are prepared from the primordial germ cells obtained fromfetuses of about 8-11 weeks of gestation (in the case of a human fetus)using laboratory techniques known to anyone skilled in the arts. Thegenital ridges are dissociated and cut into small chunks which arethereafter disaggregated into cells by mechanical dissociation. The EGcells are then grown in tissue culture flasks with the appropriatemedium. The cells are cultured with daily replacement of medium until acell morphology consistent with EG cells is observed, typically after7-30 days or 1-4 passages. For additional details on methods ofpreparation human EG cells see Shamblott et al., Proc. Natl. Acad. Sci.USA 95: 13726, 1998 and U.S. Pat. No. 6,090,622.

It will be appreciated that embryonic stem cells in an undifferentiatedstate are of a distinct morphology, which is clearly distinguishable bythe skilled in the art from that of differentiated cells of embryo oradult origin. Typically, undifferentiated embryonic stem cells have highnuclear/cytoplasmic ratios, prominent nucleoli and compact colonyformation with poorly discernable cell junctions. Additional features ofthe undifferentiated state of the embryonic stem cells are furtherdescribed hereinunder.

As used herein the phrase “expanding embryonic stem cells” refers toobtaining a plurality of embryonic stem cells from a single or apopulation of embryonic stem cells. Preferably, expanding embryonic stemcells refers also to increasing the number of embryonic stem cells overthe culturing period. It will be appreciated that the number of cellswhich can be obtained from a single embryonic stem cell depends on theproliferation capacity of the cell. The proliferation capacity of anembryonic stem cell can be calculated by the doubling time of the cell(i.e., the time needed for a cell to undergo a mitotic division in theculture) and the period the stem cell can be maintained in theundifferentiated state while in culture (which is equivalent to thenumber of passages multiplied by the days between each passage).

For example, as described in Example 2 of the Examples section whichfollows, hESCs could be maintained in the suspension culture of thepresent invention for at least 80 days while being subjected to 17serial passaging (culture splitting) which occurred every 4-6 days.Given that the hESCs cultured in suspension exhibited a doubling time of36 hours (e.g., when cultured on the CM100F medium), a single hESCcultured under these conditions could be expanded to give rise to 2⁴⁵hESCs (i.e., 3.5×10¹³ hESCs).

As mentioned, the method according to this aspect of the presentinvention is effected by culturing the embryonic stem cells in asuspension culture under culturing conditions devoid of substrateadherence and which allow expansion of the embryonic stem cells in theundifferentiated state.

As used herein the phrase “suspension culture” refers to a culture inwhich the embryonic stem cells are suspended in a medium rather thanadhering to a surface.

Thus, the culture of the present invention is “devoid of substrateadherence” in which the embryonic stem cells are capable of expandingwithout adherence to an external substrate such as components ofextracellular matrix, a glass microcarrier or beads.

Culturing according to this aspect of the present invention is effectedby plating the stem cells in a culture vessel at a cell density whichpromotes cell survival and proliferation but limits differentiation.Typically, a plating density of between about 5×10⁴-2×10⁵ cells per mlis used. It will be appreciated that although single-cell suspensions ofstem cells are usually seeded, small clusters such as 10-200 cells mayalso be used.

In order to provide the ESCs with sufficient and constant supply ofnutrients and growth factors while in the suspension culture, theculture medium can be replaced on a daily basis, or, at a pre-determinedschedule such as every 2-3 days. For example, replacement of the culturemedium can be performed by subjecting the ESC suspension culture tocentrifugation for about 3 minutes at 80 g, and resuspension of theformed ESC pellet in a fresh medium. Additionally or alternatively, aculture system in which the culture medium is subject to constantfiltration or dialysis so as to provide a constant supply of nutrientsor growth factors to the ESCs may be employed.

Since large clusters of ESCs may cause cell differentiation, measuresare taken to avoid large ESCs aggregates. Preferably, the formed ESCclumps are dissociated every 5-7 days and the single cells or smallclumps of cells are either split into additional culture vessels (i.e.,passaged) or remained in the same culture vessel yet with additionalculture medium. For dissociation of large ESC clumps, a pellet of ESCs(which may be achieved by centrifugation as described hereinabove) or anisolated ESC clump can be subject to enzymatic digestion and/ormechanical dissociation.

Enzymatic digestion of ESC clump(s) can be performed by subjecting theclump(s) to an enzyme such as type IV Collagenase (Worthingtonbiochemical corporation, Lakewood, N.J., USA) and/or Dispase (InvitrogenCorporation products, Grand Island N.Y., USA). The time of incubationwith the enzyme depends on the size of cell clumps present in thesuspension culture. Typically, when hESC cell clumps are dissociatedevery 5-7 days while in the suspension culture, incubation of 20-60minutes with 1.5 mg/ml type IV Collagenase results in small cell clumpswhich can be further cultured in the undifferentiated state.Alternatively, ESC clumps can be subjected to incubation of about 25minutes with 1.5 mg/ml type IV Collagenase followed by five minutesincubation with 1 mg/ml Dispase, essentially as described under “GeneralMaterials and Experimental Methods” of the Examples section whichfollows. It should be noted that passaging of human ESCs with trypsinmay result in chromosomal instability and abnormalities (see forexample, Mitalipova M M., et al., Nature Biotechnology, 23: 19-20, 2005and Cowan C A et al., N. Engl. J. of Med. 350: 1353-1356, 2004), andtherefore should be avoided.

Mechanical dissociation of large ESC clumps can be performed using adevice designed to break the clumps to a predetermined size. Such adevice can be obtained from CellArtis Goteborg, Sweden. Additionally oralternatively, mechanical dissociation can be manually performed using aneedle such as a 27 g needle (BD Microlance, Drogheda, Ireland) whileviewing the clumps under an inverted microscope.

Preferably, following enzymatic or mechanical dissociation of the largecell clumps, the dissociated ESC clumps are further broken to smallclumps using 200 μl Gilson pipette tips (e.g., by pipetting up and downthe cells).

The culture vessel used for culturing the ESC in suspension according tothe method of this aspect of the present invention can be any tissueculture vessel (e.g., with a purity grade suitable for culturing ESCs)having an internal surface designed such that ESC cultured therein areunable to adhere or attach to such a surface (e.g., non-tissue culturetreated cells, to prevent attachment or adherence to the surface).Preferably, in order to obtain a scalable culture, culturing accordingto this aspect of the present invention is effected using a controlledculturing system (preferably a computer-controlled culturing system) inwhich culture parameters such as temperature, agitation, pH, and pO₂ isautomatically performed using a suitable device. Once the cultureparameters are recorded, the system is set for automatic adjustment ofculture parameters as needed for ESCs expansion.

It will be appreciated that culturing according to the method of thisaspect of the present invention can be performed under dynamicconditions (i.e., under conditions in which the ESCs are subject toconstant movement while in the suspension culture) or under non-dynamicconditions (i.e., a static culture). For non-dynamic culturing of ESCs,the ESCs can be cultured in uncoated 58 mm petri dishes (Greiner,Frickenhausen, Germany). For dynamic culturing of ESCs, the ESCs can becultured in spinner flasks [e.g., of 200 ml to 1000 ml, for example 250ml which can be obtained from CellSpin of Integra Biosciences, Fernwald,Germany; of 100 ml which can be obtained from Bellco, Vineland, N.J.; orin 125 ml Erlenmeyer (Corning Incorporated, Corning N.Y., USA)] whichcan be connected to a control unit and thus present a controlledculturing system.

The medium used to culture the ESCs in suspension according to themethod of this aspect of the present invention can be any culture mediumcapable of supporting the growth of ESCs while maintaining them in anundifferentiated state. Such a culture medium can be a water-basedmedium which includes a combination of substances such as salts,nutrients, minerals, vitamins, amino acids, nucleic acids, proteins suchas cytokines, growth factors and hormones, all of which are needed forcell proliferation and are capable of maintaining the ESCs in anundifferentiated state. For example, a culture medium according to thisaspect of the present invention can be a synthetic tissue culture mediumsuch as Ko-DMEM (Gibco-Invitrogen Corporation products, Grand Island,N.Y., USA), DMEM/F12 (Biological Industries, Biet Haemek, Israel), MabADCB medium (HyClone, Utah, USA) or DMEM/F12 (Biological Industries,Biet Haemek, Israel) supplemented with the necessary additives as isfurther described hereinunder. Preferably, all ingredients included inthe culture medium of the present invention are substantially pure, witha tissue culture grade.

Preferably, in order to obtain a well-defined, xeno-free ESC culturewhich can be easily scalable and is suitable for both cell based-therapyand use in the pharmaceutical industry (e.g., for drug screening,identification of drug targets and cell-based compound delivery), theculture medium used by the method of this aspect of the presentinvention should be well-defined (i.e., with known and constantcomponents) and xeno-free (i.e., devoid of xeno contaminants).

Preferably, the culture medium used by the method of this aspect of thepresent invention is serum-free, serum replacement-free, xeno-free,feeder-free (i.e., devoid of feeder cells) and protein carrier-free.

Serum or serum replacement are usually added to most culture media whichare designed for culturing stem cells, and particularly, embryonic stemcells, in order to provide the cells with the optimal environment,similar to that present in vivo (i.e., within the organism from whichthe cells are derived, e.g., a blastocyst of an embryo or an adulttissue of a postnatal individual). However, while the use of serum whichis derived from either an animal source (e.g., bovine serum) or a humansource (human serum) is limited by the significant variations in serumcomponents between individuals and the risk of having xeno contaminants(in case of an animal serum is used), the use of the more definedcomposition such as the currently available serum Replacement™(Gibco-Invitrogen Corporation, Grand Island, N.Y. USA) may be limited bythe presence of Albumax (Bovine serum albumin enriched with lipids)which is from an animal source within the composition (InternationalPatent Publication No. WO 98/30679 to Price, P. J. et al).

A protein carrier refers to a protein which acts in the transfer ofproteins or nutrients (e.g., minerals such as zinc) to the cells in theculture. Such protein carriers can be, for example, albumin (e.g.,bovine serum albumin), Albumax (lipid enriched albumin) or plasmanate(human plasma isolated proteins). Since these carriers are derived fromeither human or animal sources their use in hESCs cultures is limited bybatch-specific variations and/or exposure to pathogens. On the otherhand, the recombinant human albumin, which is substantially pure anddevoid of animal contaminants is highly expensive, thus not commonlyused in hESCs cultures. Thus, a culture medium which is devoid of aprotein carrier is highly advantageous since it enables a truly definedmedium that can be manufacture from recombinant or synthetic materials.

Preferably, a culture medium which is serum-free, serumreplacement-free, xeno-free, feeder-free and protein carrier-free can bea culture medium which comprises a TGFβ isoform (for non-limitingexamples see the D1, D2, HA16 or HA19 culture media which are describedin Examples 1 and 2 of the Examples section which follows).

As used herein the phrase “TGFβ isoform” refers to any isoform of thetransforming growth factor beta (β) including TGFβ₁ (e.g., Homo sapiensTGFβ1, GenBank Accession No. NP_000651), TGFβ2 (e.g., Homo sapiensTGFβ2, GenBank Accession No. NP_003229) and TGFβ3 (e.g., Homo sapiensTGFβ3, GenBank Accession No. NP_003230) which function through the samereceptor signaling system in the control of proliferation,differentiation, and other functions in many cell types. TGFβ acts ininducing transformation and also acts as a negative autocrine growthfactor. According to preferred embodiments of the present invention theTGFβ isoform which is included in the culture medium of the presentinvention is TGFβ₁ or TGFβ₃. Such TGFβ isoforms can be obtained fromvarious commercial sources such as R&D Systems Minneapolis Minn., USA.

As described in Example 2 of the Examples section which follows, thepresent inventors have used various culture media which contain TGFβ₁(e.g., the D1 medium which contains 0.12 ng/ml TGFβ₁) or TGFβ₃ (e.g.,the D2 medium, the HA16 medium or the HA19 medium which contain 2 ng/mlTGFβ₃) to successfully culture hESCs in a suspension culture andmaintain them in the undifferentiated state.

Preferably, TGFβ₁ which is included in the culture medium of this aspectof the present invention is present at a concentration of at least 0.06ng/ml, more preferably, at least 0.07 ng/ml, more preferably, at least0.08 ng/ml, more preferably, at least 0.09 ng/ml, more preferably, atleast 0.1 ng/ml, more preferably, at least 0.11 ng/ml, even morepreferably, at least 0.12 ng/ml.

Preferably, TGFβ₃ which is included in the culture medium of this aspectof the present invention is present at a concentration of at least 0.5ng/ml, more preferably, at least 0.6 ng/ml, more preferably, at least0.8 ng/ml, more preferably, at least 0.9 ng/ml, more preferably, atleast 1 ng/ml, more preferably, at least 1.2 ng/ml, more preferably, atleast 1.4 ng/ml, more preferably, at least 1.6 ng/ml, more preferably,at least 1.8 ng/ml, even more preferably, at least 2 ng/ml.

Preferably, the TGFβ-containing culture medium of this aspect of thepresent invention further includes basic fibroblast growth factor(bFGF). bFGF can be obtained from any commercial supplier of tissueculture ingredients such as Invitrogen Corporation products, GrandIsland N.Y., USA.

Preferably, the bFGF which is included in TGFβ-containing culture mediumof this aspect of the present invention is present at a concentration ofat least 2 ng/ml, at least 3 ng, at least 4 ng/ml, at least 5 ng/ml, atleast 6 ng/ml, at least 7 ng, at least 8 ng/ml, at least 9 ng/ml, atleast 10 ng/ml.

Alternatively, a culture medium which is based on the IL6RIL6 chimeraand is serum or serum replacement-free, xeno-free and proteincarrier-free can be also used along with the method of this aspect ofthe present invention.

As used herein the term “IL6RIL6” refers to a chimeric polypeptide whichcomprises the soluble portion of interleukin-6 receptor (IL-6-R, e.g.,the human IL-6-R as set forth by GenBank Accession No. AAH89410 (SEQ IDNO: 32)) (e.g., a portion of the soluble IL6 receptors as set forth bySEQ ID NO: 33 which corresponds to amino acids 112-355 of GenBankAccession No. AAH89410 (SEQ ID NO: 32)) and the interleukin-6 (IL6)(e.g., human IL-6 as set forth by GenBank Accession No. CAG29292) or abiologically active fraction thereof (e.g., a receptor binding domain).Preferably, the IL6RIL6 chimera used by the method according to thisaspect of the present invention is capable of supporting theundifferentiated growth of human embryonic stem cells, while maintainingtheir pluripotent capacity. It will be appreciated that whenconstructing the IL6RIL6 chimera the two functional portions (i.e., theIL6 and its receptor) can be directly fused (e.g., attached ortranslationally fused, i.e., encoded by a single open reading frame) toeach other or conjugated (attached or translationally fused) via asuitable linker (e.g., a polypeptide linker). Preferably, the IL6RIL6chimeric polypeptide exhibits a similar amount and pattern ofglycosylation as the naturally occurring IL6 and IL6 receptor. Forexample, a suitable IL6RIL6 chimera is as set forth in SEQ ID NO:31 andin FIG. 11 of WO 99/02552 to Revel M., et al., which is fullyincorporated herein by reference.

Preferably, the IL6RIL6 chimera which is included in the culture mediumof this aspect of the present invention is present at a concentration ofat least 25 ng/ml, preferably at least 50 ng/ml, preferably, at least100 ng/ml, preferably, at least 200 ng/ml, preferably, at least 300ng/ml. It should be noted that the concentration of the IL6RIL6 chimeracan vary depending on the purity of the chimeric polypeptide followingits synthesis or recombinant expression and those of skills in the artare capable of adjusting the optimal concentration depending on suchpurity.

Preferably, the IL6RIL6-containing culture medium of this aspect of thepresent invention includes at least 2 ng/ml bFGF, at least 3 ng/ml, atleast 4 ng/ml, at least 5 ng/ml, at least 6 ng/ml, at least 7 ng, atleast 8 ng/ml, at least 9 ng/ml, at least 10 ng/ml bFGF.

For example, a suitable IL6RIL6-containing culture medium which can beused for culturing the ESC in a suspension culture can be the HACM100culture medium described under the “General Materials and ExperimentalMethods” and Example 2 of the Examples section which follows, which wasshown capable of maintaining hESCs in an undifferentiated state for atleast 1-2 passages.

Still alternatively, a culture medium which is based on solubleinterleukin-6 receptor (sIL6R) [e.g., GenBank Accession No. NM_000565.2,NM_181359.1, NP_000556.1, NP_852004.1] and soluble interleukin-6 (IL6)[e.g., GenBank Accession No. NM_000600.1, NP_000591.1] (separately) canbe also used along with the method of the present invention. Forexample, as described in Example 4 of the Examples sections whichfollows, a culture medium such as the yFIL25 which comprises 25 ng IL6and 25 ng sIL6R can be used to culture, expand and maintain human ESCsin a pluripotent, proliferative and undifferentiated state for at least19 passages. Thus, human ESCs cultured in such a culture mediumexpressed markers characteristics of the undifferentiated state,exhibited normal chromosomal karyotype (as tested after 14 passages) andwere capable of forming EBs which included all three embryonic germlayers (pluripotent). Preferably, the sIL6R is present at aconcentration of at least 10 nanogram per milliliter (ng/ml), morepreferably, at least 15 ng/ml, more preferably, at least 20 ng/ml, e.g.,at least 22 ng/ml, 25 ng/ml, 27 ng/ml, or 30 ng/ml. For example, sIL6Rcan be present at a concentration of 15-30 ng/ml, e.g., 25 ng/ml. sIL6Rand IL6 can be obtained, for example, from R&D systems, Minneapolis,Minn., USA.

Still alternatively, a culture medium which is based on leukemiainhibitory factor (LIF) [e.g., GenBank Accession No. NM_002309.2 (mRNA)or NP_002300.1 (protein)] can be also used along with the method of thepresent invention. For example, as described in Example 4 of theExamples sections which follows, a culture medium such as the yFL1,yFL2, or yFL3 can be used to culture, expand and maintain human ESCs ina pluripotent, proliferative and undifferentiated state for at least 18passages. Thus, human ESCs cultured in such a culture medium expressedmarkers characteristics of the undifferentiated state, exhibited normalchromosomal karyotype (as tested after 14 passages) and were capable offorming EBs which included all three embryonic germ layers(pluripotent). Preferably, LIF is present at a concentration of at least1000 units/ml, more preferably, at least 2000 units/ml, more preferably,at least 3000 units/ml. Human recombinant leukemia inhibitory factor(hrLIF) can be obtained from R&D Systems Minneapolis Minn., USA.

Still alternatively, a culture medium which is based on leukemiainhibitory factor (LIF) [e.g., GenBank Accession No. NM_002309.2 (mRNA)or NP_002300.1 (protein)] and TGFβ1 can be used along with the method ofthe present invention. For example, as described in Example 4 of theExamples sections which follows, a culture medium such as the TLF mediumcan be used to culture, expand and maintain human ESCs in a pluripotent,proliferative and undifferentiated state for at least 31 passages. Thus,human ESCs cultured in such a culture medium expressed markerscharacteristics of the undifferentiated state, exhibited normalchromosomal karyotype (as tested after 18 passages) and were capable offorming EBs which included all three embryonic germ layers(pluripotent).

It will be appreciated that any of the proteinaceous factors used in theculture medium of the present invention (e.g., the IL6RIL6 chimera,bFGF, TGFβ1, TGFβ3, LIF, sIL6R and IL6) can be recombinantly expressedor biochemically synthesized. In addition, naturally occurringproteinaceous factors such as bFGF and TGFβ can be purified frombiological samples (e.g., from human serum, cell cultures) using methodswell known in the art.

Biochemical synthesis of the proteinaceous factors of the presentinvention (e.g., the IL6RIL6 chimera) can be performed using standardsolid phase techniques. These methods include exclusive solid phasesynthesis, partial solid phase synthesis methods, fragment condensationand classical solution synthesis.

Recombinant expression of the proteinaceous factors of the presentinvention (e.g., the IL6RIL6 chimera) can be generated using recombinanttechniques such as described by Bitter et al., (1987) Methods inEnzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol.185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al.(1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680,Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol.Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for PlantMolecular Biology, Academic Press, NY, Section VIII, pp 421-463.Specifically, the IL6RIL6 chimera can be generated as described in PCTpublication WO 99/02552 to Revel M., et al. and Chebath J., et al.,1997, which are fully incorporated herein by reference.

For example, to generate the IL6RIL6 chimera, a polynucleotide sequenceencoding the IL6RIL6 chimera (e.g., the polypeptide set forth by SEQ IDNO:31) is preferably ligated into a nucleic acid construct suitable forexpression in a host cell [i.e., a cell in which the polynucleotideencoding the polypeptide-of-choice (e.g., the IL6RIL6 chimera) isexpressed]. Preferably, to generate an IL6RIL6 chimera with the amountand pattern of glycosylation as of the naturally occurring IL6 andIL6-R, the host cell employed is a eukaryotic host cell, more preferablya mammalian host cell such as human cell or CHO cell).

For expression in mammalian cells [e.g., CHO cells, human HEK 293 cells(ATCC CRL 1573)] a number of mammalian expression vectors can be used.Examples include, but are not limited to, pcDNA3, pcDNA3.1(+/−), pGL3,pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1,pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available fromInvitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSVand pBK-CMV which are available from Strategene, pTRES which isavailable from Clontech, and their derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp205. Other exemplary vectors include pMSG, pAV009/A+, pMT010/A+,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

Various methods can be used to introduce the expression vector of thepresent invention into host cells. Such methods are generally describedin Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringsHarbor Laboratory, New York (1989, 1992), in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.(1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995),Vectors: A Survey of Molecular Cloning Vectors and Their Uses,Butterworths, Boston Mass. (1988) and Gilboa et al., [Biotechniques 4(6): 504-512, 1986] and include, for example, stable or transienttransfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Transformed cells are cultured under effective conditions, which allowfor the expression of high amounts of the recombinant polypeptide (e.g.,the IL6RIL6 chimera). Following a predetermined time in culture,recovery of the recombinant polypeptide is effected. The phrase“recovery of the recombinant polypeptide” used herein refers tocollecting the whole fermentation medium containing the polypeptide andneed not imply additional steps of separation or purification.

Thus, polypeptides of the present invention can be purified using avariety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.

The polypeptide of the present invention (e.g., the IL6RIL6 chimera) ispreferably retrieved in “substantially pure” form. As used herein, thephrase “substantially pure” refers to a purity that allows for theeffective use of the polypeptide of the present invention (e.g., theIL6RIL6 chimera) in maintaining the human embryonic stem cells in anundifferentiated state while in culture.

Although currently less preferred, other culture media which comprisethe IL6RIL6 chimera but also include serum or serum Replacement™ (e.g.,the CM100F medium or similar media with other concentrations of theIL6RIL6 chimera such as 200 or 300 ng/ml as described in the Examplessection which follows) can be used by the method of this aspect of thepresent invention. In this case the serum (e.g., human serum) or serumReplacement™ can be provided at various concentrations, such as aconcentration of at least 10%, e.g., a concentration of at least 15%, atleast 20%, at least 25% or at least 30%.

Serum Replacement™ includes albumin or albumin substitutes, amino acids,vitamins, transferrins or transferrin substitutes, antioxidants, insulinor insulin substitutes, collagen precursors and trace elements(International Patent Publication No. WO 98/30679 to Price, P. J. etal). To provide animal-free culture conditions the albumin or albuminsubstitutes are preferably derived from a human source and/or areprepared using recombinant techniques in host cells as describedhereinabove.

When cultured according to the method of this aspect of the presentinvention, embryonic stem cell growth is monitored to determine theirdifferentiation state. The differentiation state can be determined usingvarious approaches including, for example, morphological evaluation(e.g., as shown in FIGS. 5a-g ) and/or detection of the expressionpattern of typical markers of the undifferentiated state usingimmunological techniques such as flow cytometry for membrane-boundmarkers, immunohistochemistry or immunofluorescence for extracellularand intracellular markers and enzymatic immunoassay, for secretedmolecular markers. For example, immunofluorescence employed on hESCscultured according to the method of this aspect of the present inventionrevealed the expression of Oct4, stage-specific embryonic antigen (SSEA)4, the tumour-rejecting antigen (TRA)-1-60 and TRA-1-81 (FIGS. 4a-c anddata not shown). Additionally, the level of transcripts of specificundifferentiation markers (e.g., Oct 4, Nanog, Sox2 and Rex1 as shown inFIGS. 6a-d ) or differentiation markers (e.g., albumin, glucagons,α-cardiac actin, β-globulin, Flk1, AC133 and neurofilament) can bedetected using RNA-based techniques such as RT-PCR analysis and/or cDNAmicroarray analysis.

Determination of ES cell differentiation can be also effected viameasurements of alkaline phosphatase activity. Undifferentiated human EScells have alkaline phosphatase activity which can be detected by fixingthe cells with 4% paraformaldehyde and developing with the Vector Redsubstrate kit according to manufacturer's instructions (VectorLaboratories, Burlingame, Calif., USA).

Preferably, the embryonic stem cells cultured in any of the suspensionculture media described hereinabove exhibit normal chromosomal karyotypefollowing at least 1 passage, preferably, following at least 2 passages,preferably, following at least 3 passages, preferably, following atleast 4 passages, preferably, following at least 5 passages, preferably,following at least 7 passages, preferably, following at least 10passages, preferably, following at least 12 passages, preferably,following at least 15 passages, preferably, following at least 20passages, preferably, following at least 25 passages, preferably,following at least 30 passages (e.g., hESCs exhibited normal karyotypefollowing at least 14, 18, 23 or 36 passages), thus representinggenetically stable human ESC lines.

Preferably, the embryonic stem cells cultured in any of the suspensionculture media described hereinabove exhibit a doubling time of at least20 hours, more preferably, a doubling time which is between 20 to 40hours (e.g., about 36 hours), thus representing a non-tumorigenic,genetically stable human ESCs.

It should be noted that the present invention provides, for the firsttime, a cell culture which comprises embryonic stem cells and a culturemedia which comprises the soluble interleukin-6 receptor (sIL6R) andinterleukin-6 (IL6) (separately) wherein the soluble IL6R is present ata concentration of at least 10 (ng/ml) (e.g., 25 ng/ml), and whereas theculture medium being capable of maintaining the embryonic stem cells inan undifferentiated state for at least 5 passages (see Example 4 of theExamples section which follows, which demonstrates undifferentiatedhESCs following 19 passages).

Similarly, the present invention provides, for the first time, a cellculture which comprises human embryonic stem cells and a culture mediawhich comprises LIF (at a concentration of at least 1000 u/ml), whereinthe culture medium being capable of maintaining the human ESCs in anundifferentiated state for at least 18 passages (see Example 4 of theExamples section which follows).

It will be appreciated, that the newly defined suspension culturedescribed hereinabove can be also used to derive new hESC lines in acomplete, xeno-free, scalable culture system.

Thus, according to another aspect of the present invention there isprovided a method of deriving an embryonic stem cell line. The method iseffected by (a) obtaining an embryonic stem cell from a pre-implantationstage blastocyst, post-implantation stage blastocyst and/or a genitaltissue of a fetus; and (b) culturing the embryonic stem cell in asuspension culture, under culturing conditions which allow expansion ofthe embryonic stem cells in an undifferentiated state, thereby derivingthe embryonic stem cell line.

The term “deriving” as used herein refers to generating an embryonicstem cell line from at least one embryonic stem cell.

As used herein the phrase “embryonic stem cell line” refers to embryonicstem cells which are derived from a single or a group of embryonic stemcells of a single organism (e.g., a single human blastocyst), and whichare characterized by the ability to proliferate in culture whilemaintaining the undifferentiated state and the pluripotent capacity.

Obtaining an embryonic stem cell from a pre-implantation stageblastocyst, post-implantation stage blastocyst and/or a genital tissueof a fetus can be performed using methods known in the art, as describedhereinabove and in the “General Materials and Experimental Methods” ofthe Examples section which follows. Briefly, the zona pellucida isremoved from a 5-7 day-old blastocyst using Tyrode's acidic solution(Sigma, St Louis Mo., USA), the trophoblast layer is specificallyremoved either by immunosurgery or mechanically using 27 g needles andthe exposed ICM is either directly cultured in a suspension culture witha suitable culture medium (e.g., the CM100F, HA16 or D2 medium) for 4-10days (in case a preimplantation blastocyst is used) or subject to invitro implantation by culturing the ICM for 6-8 days (to obtain cells ofa 13 day-old blastocyst in case a post-implantation/pre-gastrulationblastocyst is used) on feeder layers or a feeder-free culturing systemwhich allow implantation of the blastocyst to the surface, followingwhich the implanted cells are isolated and further cultured insuspension as described hereinunder. When using the genital tissue of afetus, the genital ridges are dissociated and cut into small chunkswhich are thereafter disaggregated into cells by mechanicaldissociation. The single cell EG cells are then cultured in suspensionculture with a suitable culture medium (e.g., the CM100F, HA16 or D2medium) for 4-10 days.

Once obtained the ESCs are further cultured in suspension underconditions which allow expansion of the embryonic stem cells in theundifferentiated state, essentially as described hereinabove.

Preferably, the cell culture of the present invention is characterizedby at least 40%, at least 50%, at least 60%, more preferably at least70%, more preferably at least 80%, most preferably at least 85% ofundifferentiated embryonic stem cells.

It will be appreciated that an established embryonic stem cell line canbe subject to freeze/thaw cycles without hampering the proliferativecapacity of the cells in the undifferentiated state while preservingtheir pluriptent capacity. For example, as is shown in the Examplessection which follows, using 15% SR and 10% DMSO, hESCs weresuccessfully frozen and thawed.

As described in Example 2 of the Examples section which follows, hESCswhich were expanded and maintained in the suspension culture describedhereinabove are pluripotent (i.e., capable of differentiating into allcell types of the three embryonic germ layers, the ectoderm, theendoderm and the mesoderm) as evidenced in vitro (by the formation ofEBs). Thus, hESCs cultured according to the teachings of the presentinvention can be used as a source for generating differentiated,lineage-specific cells. Such cells can be obtained directly from theESCs by subjecting the ESCs to various differentiation signals (e.g.,cytokines, hormones, growth factors) or indirectly, via the formation ofembryoid bodies and the subsequent differentiation of cells of the EBsto lineage-specific cells.

Thus, according to yet an additional aspect of the present inventionthere is provided a method of generating embryoid bodies from embryonicstem cells. The method is effected by (a) culturing the embryonic stemcells in a suspension culture under culturing conditions which allowexpansion of the embryonic stem cells in an undifferentiated state tothereby obtain expanded, undifferentiated embryonic stem cells; and (b)subjecting the expanded, undifferentiated embryonic stem cells toculturing conditions suitable for differentiating the embryonic stemcells to embryoid bodies; thereby generating the embryoid bodies fromthe embryonic stem cells.

As used herein the phrase “embryoid bodies” refers to morphologicalstructures comprised of a population of ESCs, extended blastocyst cells(EBCs) and/or embryonic germ cells (EGCs) which have undergonedifferentiation. EBs formation initiates following the removal ofdifferentiation blocking factors from ES cell cultures. In the firststep of EBs formation, ESCs proliferate into small masses of cells whichthen proceed with differentiation. In the first phase ofdifferentiation, following 1-4 days in culture for human ESCs, a layerof endodermal cells is formed on the outer layer of the small mass,resulting in “simple EBs”. In the second phase, following 3-20 dayspost-differentiation, “complex EBs” are formed. Complex EBs arecharacterized by extensive differentiation of ectodermal and mesodermalcells and derivative tissues.

Thus, the method of this aspect of the present invention involves theculturing of ESCs in a suspension culture using any of the culture mediadescribed hereinabove in order to obtain expanded, undifferentiatedembryonic stem cells and then subjecting the expanded, undifferentiatedESCs to culturing conditions suitable for differentiating the ESCs toembryoid bodies. Such culturing conditions are substantially devoid ofdifferentiation inhibitory factors which were employed during step (a),e.g., a TGFβ isoform or the IL6RIL6 chimera.

For EBs formation, the ESCs are transferred from the suspension cultureswhich include a culture medium capable of maintaining the ESCs in anundifferentiated state to a suspension culture in the presence of aculture medium containing serum or serum replacement and being devoid ofdifferentiation-inhibitory factors, essentially as described in the“General Materials and Experimental Methods” of the Examples sectionwhich follows. For example, a culture medium suitable for EBs formationmay include a basic culture medium (e.g., Ko-DMEM or DMEM/F12)supplemented with 20% FBSd (HyClone, Utah, USA), 1 mM L-glutamine, 0.1mM β-mercaptoethanol, and 1% non-essential amino acid stock.

Monitoring the formation of EBs can be effected by morphologicalevaluations (e.g., histological staining as described in Example 2) anddetermination of expression of differentiation-specific markers [usinge.g., immunological techniques or RNA-based analysis (e.g., RT-PCR, cDNAmicroarray)]. Non-limiting examples of differentiation-specific markersof all three embryonic germ layers include albumin and glucagon (typicalof the embryonic endoderm), α-cardiac actin, β-globulin and Flk1(typical of the embryonic mesoderm), and AC133 and neurofilament (NFH)(typical of the embryonic ectoderm).

It will be appreciated that in order to obtain lineage-specific cellsfrom the EBs, cells of the EBs can be further subjected to culturingconditions suitable for lineage-specific cells.

Preferably, the method of this aspect of the present invention furtherincludes step (c): subjecting cells of the embryoid bodies to culturingconditions suitable for differentiating and/or expanding lineagespecific cells; thereby generating the lineage-specific cells from theembryonic stem cells.

As used herein the phrase “culturing conditions suitable fordifferentiating and/or expanding lineage specific cells” refers to acombination of a culture system, e.g., feeder cell layers, feeder-freematrix or a suspension culture and a culture medium which are suitablefor the differentiation and/or expansion of specific cell lineagesderived from cells of the EBs. Non-limiting examples of such culturingconditions are further described hereinunder.

Preferably, the method of this aspect of the present invention furtherincludes isolating lineage specific cells following step (b).

As used herein, the phrase “isolating lineage specific cells” refers tothe enrichment of a mixed population of cells in a culture with cellspredominantly displaying at least one characteristic associated with aspecific lineage phenotype. It will be appreciated that all celllineages are derived from the three embryonic germ layers. Thus, forexample, hepatocytes and pancreatic cells are derived from the embryonicendoderm, osseous, cartilaginous, elastic, fibrous connective tissues,myocytes, myocardial cells, bone marrow cells, vascular cells (namelyendothelial and smooth muscle cells), and hematopoietic cells aredifferentiated from embryonic mesoderm and neural, retina and epidermalcells are derived from the embryonic ectoderm.

According to one preferred embodiment of the present invention,isolating is effected by sorting of cells of the EBs via fluorescenceactivated cell sorter (FACS).

Methods of isolating EB-derived-differentiated cells via FACS analysisare known in the art. According to one method, EBs are disaggregatedusing a solution of Trypsin and EDTA (0.025% and 0.01%, respectively),washed with 5% fetal bovine serum (FBS) in phosphate buffered saline(PBS) and incubated for 30 minutes on ice with fluorescently-labeledantibodies directed against cell surface antigens characteristics to aspecific cell lineage. For example, endothelial cells are isolated byattaching an antibody directed against the platelet endothelial celladhesion molecule-1 (PECAM1) such as the fluorescently-labeled PECAM1antibodies (30884X) available from PharMingen (PharMingen, BectonDickinson Bio Sciences, San Jose, Calif., USA) as described inLevenberg, S. et al., (Endothelial cells derived from human embryonicstem cells. Proc. Natl. Acad. Sci. USA. 2002. 99: 4391-4396).Hematopoietic cells are isolated using fluorescently-labeled antibodiessuch as CD34-FITC, CD45-PE, CD31-PE, CD38-PE, CD90-FITC, CD117-PE,CD15-FITC, class I-FITC, all of which IgG1 are available fromPharMingen, CD133/1-PE (IgG1) (available from Miltenyi Biotec, Auburn,Calif.), and glycophorin A-PE (IgG1), available from Immunotech (Miami,Fla.). Live cells (i.e., without fixation) are analyzed on a FACScan(Becton Dickinson Bio Sciences) by using propidium iodide to excludedead cells with either the PC-LYSIS or the CELLQUEST software. It willbe appreciated that isolated cells can be further enriched usingmagnetically-labeled second antibodies and magnetic separation columns(MACS, Miltenyi) as described by Kaufman, D. S. et al., (Hematopoieticcolony-forming cells derived from human embryonic stem cells. Proc.Natl. Acad. Sci. USA. 2001, 98: 10716-10721).

According to yet an additional preferred embodiment of the presentinvention, isolating is effected by a mechanical separation of cells,tissues and/or tissue-like structures contained within the EBs.

For example, beating cardiomyocytes can be isolated from EBs asdisclosed in U.S. Pat. Appl. No. 20030022367 to Xu et al. Four-day-oldEBs of the present invention are transferred to gelatin-coated plates orchamber slides and are allowed to attach and differentiate.Spontaneously contracting cells, which are observed from day 8 ofdifferentiation, are mechanically separated and collected into a 15-mLtube containing low-calcium medium or PBS. Cells are dissociated usingCollagenase B digestion for 60-120 minutes at 37° C., depending on theCollagenase activity. Dissociated cells are then resuspended in adifferentiation KB medium (85 mM KCl, 30 mM K₂HPO₄, 5 mM MgSO₄, 1 mMEGTA, 5 mM creatine, 20 mM glucose, 2 mM Na₂ATP, 5 mM pyruvate, and 20mM taurine, buffered to pH 7.2, Maltsev et al., Circ. Res. 75:233, 1994)and incubated at 37° C. for 15-30 minutes. Following dissociation cellsare seeded into chamber slides and cultured in the differentiationmedium to generate single cardiomyocytes capable of beating.

According to still additional preferred embodiments of the presentinvention, isolating is effected by subjecting the EBs todifferentiation factors to thereby induce differentiation of the EBsinto lineage specific differentiated cells.

Following is a non-limiting description of a number of procedures andapproaches for inducing differentiation of EBs to lineage specificcells.

To differentiate the EBs of the present invention into neuralprecursors, four-day-old EBs are cultured for 5-12 days in tissueculture dishes including DMEM/F-12 medium with 5 mg/ml insulin, 50 mg/mltransferrin, 30 nM selenium chloride, and 5 mg/ml fibronectin (ITSFnmedium, Okabe, S. et al., 1996, Mech. Dev. 59: 89-102). The resultantneural precursors can be further transplanted to generate neural cellsin vivo (Brüstle, 0. et al., 1997. In vitro-generated neural precursorsparticipate in mammalian brain development. Proc. Natl. Acad. Sci. USA.94: 14809-14814). It will be appreciated that prior to theirtransplantation, the neural precursors are trypsinized and triturated tosingle-cell suspensions in the presence of 0.1% DNase.

EBs of the present invention can differentiate to oligodendrocytes andmyelinate cells by culturing the cells in modified SATO medium, i.e.,DMEM with bovine serum albumin (BSA), pyruvate, progesterone,putrescine, thyroxine, triiodothryonine, insulin, transferrin, sodiumselenite, amino acids, neurotrophin 3, ciliary neurotrophic factor andHepes (Bottenstein, J. E. & Sato, G. H., 1979, Proc. Natl. Acad. Sci.USA 76, 514-517; Raff, M. C., Miller, R. H., & Noble, M., 1983, Nature303: 390-396]. Briefly, EBs are dissociated using 0.25% Trypsin/EDTA (5min at 37° C.) and triturated to single cell suspensions. Suspendedcells are plated in flasks containing SATO medium supplemented with 5%equine serum and 5% fetal calf serum (FCS). Following 4 days in culture,the flasks are gently shaken to suspend loosely adhering cells(primarily oligodendrocytes), while astrocytes are remained adhering tothe flasks and further producing conditioned medium. Primaryoligodendrocytes are transferred to new flasks containing SATO mediumfor additional two days. Following a total of 6 days in culture,oligospheres are either partially dissociated and resuspended in SATOmedium for cell transplantation, or completely dissociated and a platedin an oligosphere-conditioned medium which is derived from the previousshaking step [Liu, S. et al., (2000). Embryonic stem cells differentiateinto oligodendrocytes and myelinate in culture and after spinal cordtransplantation. Proc. Natl. Acad. Sci. USA. 97: 6126-6131].

For mast cell differentiation, two-week-old EBs of the present inventionare transferred to tissue culture dishes including DMEM mediumsupplemented with 10% FCS, 2 mM L-glutamine, 100 units/ml penicillin,100 mg/ml streptomycin, 20% (v/v) WEHI-3 cell-conditioned medium and 50ng/ml recombinant rat stem cell factor (rrSCF, Tsai, M. et al., 2000. Invivo immunological function of mast cells derived from embryonic stemcells: An approach for the rapid analysis of even embryonic lethalmutations in adult mice in vivo. Proc Natl Acad Sci USA. 97: 9186-9190).Cultures are expanded weekly by transferring the cells to new flasks andreplacing half of the culture medium.

To generate hemato-lymphoid cells from the EBs of the present invention,2-3 days-old EBs are transferred to gas-permeable culture dishes in thepresence of 7.5% CO₂ and 5% 02 using an incubator with adjustable oxygencontent. Following 15 days of differentiation, cells are harvested anddissociated by gentle digestion with Collagenase (0.1 unit/mg) andDispase (0.8 unit/mg), both are available from F. Hoffman—La Roche Ltd,Basel, Switzerland. CD45-positive cells are isolated using anti-CD45monoclonal antibody (mAb) M1/9.3.4.HL.2 and paramagnetic microbeads(Miltenyi) conjugated to goat anti-rat immunoglobulin as described inPotocnik, A. J. et al., (Immunology Hemato-lymphoid in vivoreconstitution potential of subpopulations derived from in vitrodifferentiated embryonic stem cells. Proc. Natl. Acad. Sci. USA. 1997,94: 10295-10300). The isolated CD45-positive cells can be furtherenriched using a single passage over a MACS column (Miltenyi).

It will be appreciated that the culturing conditions suitable for thedifferentiation and expansion of the isolated lineage specific cellsinclude various tissue culture media, growth factors, antibiotic, aminoacids and the like and it is within the capability of one skilled in theart to determine which conditions should be applied in order to expandand differentiate particular cell types and/or cell lineages.

Additionally or alternatively, lineage specific cells can be obtained bydirectly inducing the expanded, undifferentiated ESCs to culturingconditions suitable for the differentiation of specific cell lineage.

In addition to the lineage-specific primary cultures, EBs of the presentinvention can be used to generate lineage-specific cell lines which arecapable of unlimited expansion in culture.

Cell lines of the present invention can be produced by immortalizing theEB-derived cells by methods known in the art, including, for example,expressing a telomerase gene in the cells (Wei, W. et al., 2003. MolCell Biol. 23: 2859-2870) or co-culturing the cells with NIH 3T3hph-HOX11 retroviral producer cells (Hawley, R. G. et al., 1994.Oncogene 9: 1-12).

It will be appreciated that since the lineage-specific cells or celllines obtained according to the teachings of the present invention aredeveloped by differentiation processes similar to those naturallyoccurring in the human embryo they can be further used for humancell-based therapy and tissue regeneration.

Thus, the present invention envisages the use of the expanded and/ordifferentiated lineage-specific cells or cell lines of the presentinvention for treating a disorder requiring cell replacement therapy.

For example, oligodendrocyte precursors can be used to treat myelindisorders (Repair of myelin disease: Strategies and progress in animalmodels. Molecular Medicine Today. 1997. pp. 554-561), chondrocytes ormesenchymal cells can be used in treatment of bone and cartilage defects(U.S. Pat. No. 4,642,120) and cells of the epithelial lineage can beused in skin regeneration of a wound or burn (U.S. Pat. No. 5,716,411).

For certain disorders, such as genetic disorders in which a specificgene product is missing [e.g., lack of the CFTR gene-product in cysticfibrosis patients (Davies J C, 2002. New therapeutic approaches forcystic fibrosis lung disease. J. R. Soc. Med. 95 Suppl 41:58-67)],ESC-derived cells are preferably manipulated to over-express the mutatedgene prior to their administration to the individual. It will beappreciated that for other disorders, the ESC-derived cells should bemanipulated to exclude certain genes.

Over-expression or exclusion of genes can be effected using knock-inand/or knock-out constructs [see for example, Fukushige, S. and Ikeda,J. E.: Trapping of mammalian promoters by Cre-lox site-specificrecombination. DNA Res 3 (1996) 73-50; Bedell, M. A., Jerkins, N. A. andCopeland, N. G.: Mouse models of human disease. Part I: Techniques andresources for genetic analysis in mice. Genes and Development 11 (1997)1-11; Bermingham, J. J., Scherer, S. S., O'Connell, S., Arroyo, E.,Kalla, K. A., Powell, F. L. and Rosenfeld, M. G.: Tst-1/Oct-6/SCIPregulates a unique step in peripheral myelination and is required fornormal respiration. Genes Dev 10 (1996) 1751-62].

In addition to cell replacement therapy, the lineage specific cells ofthe present invention can also be utilized to prepare a cDNA library.mRNA is prepared by standard techniques from the lineage specific cellsand is further reverse transcribed to form cDNA. The cDNA preparationcan be subtracted with nucleotides from embryonic fibroblasts and othercells of undesired specificity, to produce a subtracted cDNA library bytechniques known in the art.

The lineage specific cells of the present invention can be used toscreen for factors (such as small molecule drugs, peptides,polynucleotides, and the like) or conditions (such as culture conditionsor manipulation) that affect the differentiation of lineage precursor toterminally differentiated cells. For example, growth affectingsubstances, toxins or potential differentiation factors can be tested bytheir addition to the culture medium.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., Ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(Eds.) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., Ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., Ed. (1994); Stites et al.(Eds.), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (Eds.), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., Ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,Eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., Ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

General Materials and Experimental Methods

ESC culture—Human embryonic stem cell (hESC) lines I-6, I4 and I-3[Amit&Itskovitz-Eldor, 2002] were cultured with inactivated mouseembryonic fibroblasts (MEFs) for 40-60 passages in a “basic hESC culturemedium” consisting of 85% DMEM/F12 (Biological Industries, Biet Haemek,Israel) supplemented with 15% serum replacement (SR), 2 mM L-glutamine,0.1 mM β-mercaptoethanol, 1% non-essential amino acid stock, and 4 ng/mlbasic fibroblast growth factor (bFGF) (all but mentioned are from GibcoInvitrogen Corporation products, Grand Island N.Y., USA). This basicculture medium was used for the routine culture of hESCs in 2D culturewith MEFs as control.

Tested media on the feeder layer, feeder-free or suspension cultures—Thetested medium were as follows:

TGF β-Containing Media

(i) D1 medium—Mab ADCB medium (HyClone, Utah, USA) supplemented with 2mM L-glutamine (Invitrogen Corporation products, Grand Island N.Y.,USA), 0.12 ng/ml TGFβ₁ (from R&D Systems Minneapolis Minn., USA), and 10ng/ml bFGF (Invitrogen Corporation products, Grand Island N.Y., USA).

(ii) D2 medium—Mab ADCB medium (HyClone, Utah, USA) supplemented with 2mM L-glutamine (Invitrogen Corporation products, Grand Island N.Y.,USA), 2 ng/ml TGFβ₃ and 10 ng/ml bFGF (Invitrogen Corporation products,Grand Island N.Y., USA).

(iii) HA16 medium—96% DMEM/F12 (Biological Industries, Biet Haemek,Israel) supplemented with 1:1000 dilution of the ITS Premix [the ITSpremix is a ×1000 stock solution obtained from BD Biosciences, Bedford,Mass., USA and consists of 12.5 mg Insulin, 12.5 mg Transferrin and 12.5mg Selenius acid], 2 mM L-glutamine, 2 ng/ml TGFβ₃ (from R&D SystemsMinneapolis Minn., USA), 4 ng/ml bFGF, 500 ng/ml ascorbic acid (Sigma,Steinheim, Germany), and a 1:1000 dilution of a lipid mixture (SigmaCat. No. L5146, Steinheim, Germany) (all but those otherwise specifiedwere obtained from Gibco Invitrogen Corporation products, Grand IslandN.Y., USA).

(iv) HA19 medium—96% DMEM/F12 (Biological Industries, Beth Haemek,Israel) supplemented with 1:1000 dilution of the ITS premix (BDBiosciences, Bedford, Mass., USA), 2 mM L-glutamine, 2 ng/ml TGFβ₃ (fromR&D Systems Minneapolis Minn., USA), 4 ng/ml bFGF, 500 ng/ml ascorbicacid (Sigma, Steinheim, Germany), a 1:1000 dilution of a lipid mixture(Sigma Cat. No. L5146, Steinheim, Germany) and a 1:100 dilution ofSimfronic 68 (Pluronic F-68 solution, P5556 from Sigma, Steinheim,Germany, the stock is 10%, the F-68 in culture is provided at aconcentration of 0.1%) (Sigma, Steinheim, Germany) (all but thoseotherwise specified were obtained from Gibco Invitrogen Corporationproducts, Grand Island N.Y., USA).

IL6RIL6 Chimera-Containing Media

(i) CM100F medium—85% DMEM/F12 (Biological Industries, Biet Haemek,Israel) supplemented with 15% serum replacement (SR), 2 mM L-glutamine,0.1 mM β-mercaptoethanol, 1% non-essential amino acid stock, 4 ng/mlbasic fibroblast growth factor (bFGF) and 100 ng/ml IL6RIL6 chimera (akind gift from Prof. Revel M, the Weizmann Inst. Rehovot, Israel;Chebath J, et al., 1997 and WO 99/02552 to Revel M., et al. SEQ IDNO:31) (all but those otherwise specified were obtained from GibcoInvitrogen Corporation products, Grand Island N.Y., USA). As a control,the same culture media was used with the removal of the growth factors(except for bFGF which remained in the control culture medium) and theIL6RIL6 chimera.

(ii) HACM100 medium—96% DMEM/F12 (Biological Industries, Biet Haemek,Israel) supplemented with a 1:1000 dilution of the ITS premix (BDBiosciences, Bedford, Mass., USA), 2 mM L-glutamine, 4 ng/ml bFGF, 500ng/ml ascorbic acid (Sigma, Steinheim, Germany), a 1:1000 dilution of alipid mixture (Sigma Cat. No. L5146, Steinheim, Germany) and 100 ng/mlof IL6RIL6 chimera.

(iii) CM6 medium—85% Ko-DMEM (or 85% DMEM/F12, Biological Industries,Biet Haemek, Israel), supplemented with 15% serum replacement (SR), 2 mML-glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acidstock, and 4 ng/ml bFGF, 0.3 ng/ml Interleukin-6 (IL6) and 0.5 ng/ml IL6soluble receptor (both from R&D Systems Minneapolis Minn., USA) (allGibco Invitrogen Corporation products, Grand Island N.Y., USA).

(iv) “IL6-IL-6 receptor (IL6RIL6) chimera”—85% Ko-DMEM, supplementedwith 15% serum replacement (SR), 2 mM L-glutamine, 0.1 mMβ-mercaptoethanol, 1% non-essential amino acid stock, 4 ng/ml bFGF and50 ng/ml, 100 ng/ml, 200 ng/ml or 300 ng/ml of IL6RIL6 chimera (ChebathJ, et al., 1997 and WO 99/02552 to Revel M., et al. SEQ ID NO:31) (allGibco Invitrogen Corporation products, Grand Island N.Y., USA). Whenused with 100 ng/ml of the IL6RIL6 chimera, this medium is also calledCM100.

(v) Control medium—85% Ko-DMEM, supplemented with 15% serum replacement(SR), 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential aminoacid stock, 4 ng/ml bFGF (all Gibco Invitrogen Corporation products,Grand Island N.Y., USA).

Feeder Layers or Feeder-Free Culturing Systems—To test the ability ofvarious culture media to support the growth of hESC in anundifferentiated yet pluripotent state the hESCs were transferred toseveral culture systems:

(i) Fibronectin feeder-free culture system—50 μg per 10 cm²fibronectin-covered plates (human plasma fibronectin, ChemiconInternational, Temecula Calif., USA);

(ii) Matrigel™ feeder-free culture system—Matrigel™ (BD Biosciences,Bedford, Mass., USA);

(iii) MEFs—mouse embryonic fibroblast feeder layer system;

(iv) Foreskins fibroblasts—foreskin fibroblasts feeder layer system.

Passaging of hESCs on Feeder Layers or Feeder-Free CulturingSystems—Cells were passaged every four to six days using 1.5 mg/ml typeIV collagenase (Worthington biochemical corporation, Lakewood, N.J.,USA). Cells were frozen in liquid nitrogen using a freezing solutionconsisting of 10% DMSO (Sigma, St Louis Mo., USA), 40% human serum(HyClone, Utah, USA) and 50% DMEM/F12 (Biological Industries, BeitHaemek, Israel).

Culture in suspension—To examine the possibility of using theTGFβ-containing medium which is devoid of serum, serum replacement andalbumin for scalable culture of hESCs in suspension, hESCs were culturedin suspension in 58 mm petri dishes (Greiner, Frickenhausen, Germany) ina cell density of 5×10⁴-2×10⁵ cells/ml. The HA16 medium was supplementedwith 0.1% F68 (Sigma, St. Louis, Mo., USA) for the suspended culture.The culture medium in the suspension culture was changed on a dailybasis. The basic media used for culturing hESCs in suspension (which canbe further supplemented with the additive and growth factors asdescribed hereinabove) were DMEM, ko-DMEM, DMEM/F12, MabADCB or NCTCmedium.

“Passaging” of hESCs in suspension culture—The cells were passage every5-7 days using either 30-60 minute incubation with 1.5 mg/ml type IVCollagenase (Worthington biochemical corporation, Lakewood, N.J., USA)or 25 minutes incubation with 1.5 mg/ml type IV Collagenase followed byfive minutes incubation with 1 mg/ml Dispase (Invitrogen Corporationproducts, Grand Island N.Y., USA), and further broken into small clumpsusing 200 μl Gilson pipette tips. Alternatively, the cells were passagedmechanically using 27 g needles.

Following continuous culturing under these conditions the cells weretested for hESC characteristics.

RT PCR analysis—Total RNA was isolated from hESCs grown for 10-15passages in the suspension culture using Tri-Reagent (Sigma, St. LouisMo., USA), according to the manufacturer's instructions. cDNA wassynthesized from 1 μg total RNA using MMLV reverse transcriptase RNase Hminus (Promega, Madison Wis., USA). PCR reactions included denaturationfor 5 minutes at 94° C. followed by repeated cycles of 94° C. for 30seconds, annealing for 30 seconds at an annealing temperature asspecified in Table 1, hereinbelow and extension at 72° C. for 30seconds. PCR primers and reaction conditions used are described in Table1, hereinbelow. PCR products were size-fractionated using 2% agarose gelelectrophoresis. DNA markers were used to confirm the size of theresultant fragments.

TABLE 1 RT-PCR conditions Gene product (Accession Reaction Size number)Forward (F) and reverse (R) primers (SEQ ID NO:) Condition (bp) Oct-4F: 5′-GAGAACAATGAGAACCTTCAGGA (SEQ ID 30 cycles 219 (S81255) NO: 1)at 60° C. R: 5′-TTCTGGCGCCGGTTACAGAACCA (SEQ ID in 1.5 NO: 2) mM MgCl₂Albumin F: 5′-TGCTTGAATGTGCTGATGACAGGG (SEQ ID 35 cycles 302 (AF542069)NO: 3) at 60° C. R: 5′-AAGGCAAGTCAGCAGCCATCTCAT (SEQ ID in 1.5 NO: 4) mMMgCl₂ α-fetoprotein F: 5′-GCTGGATTGTCTGCAGGATGGGGAA (SEQ ID 30 cycles216 (BC027881) NO: 5) at 60° C. R: 5′-TCCCCTGAAGAAAATTGGTTAAAAT (SEQ IDin 1.5 NO: 6) mM MgCl₂ NF-68KDF: 5′-GAGTGAAATGGCACGATACCTA (SEQ ID NO: 7) 30 cycles 473 (NFHR: 5′-TTTCCTCTCCTTCTTCACCTTC (SEQ ID NO: 8) at 60° C. (AY156690; in 2 mMX15307; MgCl₂ X15309) α-cardiacF: 5′-GGAGTTATGGTGGGTATGGGTC (SEQ ID NO: 9) 35 cycles 486 actinR: 5′-AGTGGTGACAAAGGAGTAGCCA (SEQ ID at 65° C. (NM_005159) NO: 10)in 2 mM MgCl₂ β - Actin F: 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG 35 cycles838 (NM_001101) (SEQ ID NO: 11) at 62° C.R: 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC in 1.5 (SEQ ID NO: 12) mM MgCl₂Sox2 5′ CCCCCGGCGGCAATAGCA (SEQ ID NO: 13) 35 cycles 448 (Z31560) 3′TCGGCGCCGGGGAGATACAT (SEQ ID NO: 14) at 60° C. in 1.5 mM MgCl₂ Rex1 5′GCGTACGCAAATTAAAGTCCAGA (SEQ ID NO: 15) 35 cycles 306 (AF450454) 3′CAGCATCCTAAACAGCTCGCAGAAT (SEQ ID at 56° C. NO: 16) in 1.5 mM MgCl₂ CX435′ TACCATGCGACCAGTGGTGCGCT (SEQ ID NO: 17) 35 cycles 295 (NM_000165)3′GAATTCTGGTTATCATCGGGGAA (SEQ ID NO: 18) at 61° C. in 1.5 mM MgCl₂ FGF45′ CTACAACGCCTACGAGTCCTACA (SEQ ID NO: 19) 35 cycles 370 (NM_002007) 3′GTTGCACCAGAAAAGTCAGAGTTG (SEQ ID at 52° C. NO: 20) in 1.5 mM MgCl₂Glucagon 5′ CTCAGTGATCCTGATCAGATGAACG (SEQ ID 35 cycles 370 (X03991)NO: 21) at 65° C. 3′ AGTCCCTGGCGGCAAGATTATCAAG (SEQ ID in 1.5 NO: 22) mMMgCl₂ β-globulin 5′ ACCTGACTCCTGAGGAGAAGTCTGC (SEQ ID 35 cycles 410(V00499) NO: 23) at 65° C. 3′ TAGCCACACCAGCCACCACTTTCTG (SEQ ID in 1.5NO: 24) mM MgCl₂ Flk1 5′ ATGCACGGCATCTGGGAATC (SEQ ID NO: 25) 35 cycles537 (NM_002253) 3′ GCTACTGTCCTGCAAGTTGCTGTC (SEQ ID NO: 26) at 65° C.in 1.5 mM MgCl₂ AC133 5′ CAGTCTGACCAGCGTGAAAA (SEQ ID NO: 27) 35 cycles200 (NM_006017) 3′ GGCCATCCAAATCTGTCCTA (SEQ ID NO: 28) at 65° C. in 1.5mM MgCl₂ Nanog 5′ ACTAACATGAGTGTGGATCC (SEQ ID NO: 29) 35 cycles 800(NG_004095) 3′ TCATCTTCACACGTCTTCAG (SEQ ID NO: 30) at 61° C. in 1.5 mMMgCl₂ Table 1 RT-PCR primers and PCR conditions are provided along withthe GenBank Accession numbers of the amplified transcripts.

Immunohistochemistry—Undifferentiated hESCs grown in the tested culturesystem were fixed with 4% paraformaldehyde and exposed to the primaryantibodies (1:50) overnight at 4° C. Stage-specific embryonic antigen(SSEA) 1, 3 and 4 (Hybridoma Bank, Iowa, USA), tumor recognition antigen(TRA) 1-60 and TRA1-81 (Chemicon International, Temecula Calif., USA)and Oct 4 (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) were usedas primary antibodies. Cys 3 conjugated antibodies (ChemiconInternational, Temecula Calif., USA) were used as secondary antibodies(1:200 dilution).

Karyotype analysis—Karyotype analysis (G-banding) was performed on atleast 20 cells from each sample, two samples per test, as previouslydescribed [Amit et al, 2003]. Karyotypes were analyzed and reportedaccording to the “International System for Human CytogeneticNomenclature” (ISCN).

EB formation from hESCs cultured in suspension—For the formation of EBs,one to three 58 mm petri dishes (Greiner, Frickenhausen, Germany)containing ESCs in suspension cultures were transferred to new 58 mmpetri dishes containing EBs-differentiation medium consisting of 80%DMEM/F12 (Biological Industries, Beit Haemek, Israel), supplemented with20% FBSd (HyClone, Utah, USA), and 1 mM L-glutamine (InvitrogenCorporation, Grand Island N.Y., USA). Alternatively, prior to theirtransfer to the EB-differentiation medium, the ESCs were subject totreatment with 1 mg/ml type IV collagenase and further broken into smallclumps using 1000 μl Gilson pipette tips. 10 day-old EBs were harvestedfor RNA isolation and histological examination.

EB formation from hESCs cultured on 2-D (feeder free or feederlayers)—For the formation of EBs, one three confluent wells were used ina six-well plate (30 cm²). ESCs were removed from their culture dishusing 1 mg/ml type IV collagenase, further broken into small clumpsusing 1000 μl Gilson pipette tips, and cultured in suspension in 58 mmpetri dishes (Greiner, Frickenhausen, Germany). EBs were grown indifferentiation medium consisting of 80% DMEM/F12 (BiologicalIndustries, Beit Haemek, Israel), supplemented with 20% FBSd (HyClone,Utah, USA), and 1 mM L-glutamine (Invitrogen Corporation, Grand IslandN.Y., USA).

Teratoma formation—For teratoma formation, cells cultured in the offeredculture methods for more than 15 passages, were injected into the rearleg muscle of 4-week-old male SCID-beige mice (two mice for each testedculture system). Cell numbers ranged from 5×10⁶ cells to 10⁷ cells perinjection. Three to eight to 12 weeks after injection the mice weresacrificed and the resulting teratomas examined histologically.

Derivation of New hESC Lines in a Suspension Culture with the TGFβ-Containing Medium Devoid of Serum, Serum Replacement and Albumin

Blastocyst cultivation—Zygotes were donated by couples undergoingpre-implantation genetic diagnosis (PGD) or in vitro fertilization (IVF)at Cornell Medical College, NY, who signed informed consent forms. Thecouples underwent the traditional IVF procedure after ovarianstimulation with gonadotropins and oocyte retrieval. Zygotes werecultured to the blastocyst stage according to IVF laboratory standardprotocol: under oil using specialized C1/C2 media for insemination,growth and blastocyst development (Cornell).

Derivation of hESC lines in a suspension culture—Following the removalof the zona pellucida using Tyrode's acidic solution (Sigma, St LouisMo., USA), the trophoblast layer is specifically removed either byimmunosurgery or mechanically using 27 g needles. The exposed ICM isfurther cultured in suspension culture with a suitable culture medium(e.g., the CM100F, HA16 or D2) for 4-10 days. Initially, the cells aremechanically split using 27 g needles.

Derivation of hESC lines on foreskin fibroblasts—After digestion of thezona pellucida by Tyrode's acidic solution (Sigma, St Louis Mo., USA) orits mechanical removal, the exposed blastocysts were placed in whole ona mitotically inactivated foreskin fibroblasts feeder layer (line F21which was cultured in an animal free medium since its derivation untilused). For the derivation and initial passages, cells were grown in theD2 or HA16 culture medium. The cells were initially passagedmechanically every four to ten days.

Example 1 Culturing Human Embryonic Stem Cell on Feeder-Free CultureSystems with a Medium Containing TGF-Beta Isoforms and being Devoid ofSerum, Serum Replacement and Albumin

Experimental Results

In this study the ability of few medium combinations, HA16, HA19, D1,D2, and CM100 to support undifferentiated and prolonged culture of hESCsin different culture conditions was examined. The basic medium, D1 orD2, is a commercial medium design for industrial and clinical proposesfor the culture of hybridomas in suspension. The medium is free fromanimal, serum products and proteins. HA16 and HA19 are based on definedmaterials only. The CM100 medium contains the IL6RIL6 chimera and serumreplacement.

The effect of two isoforms of TGFβ, TGFβ₁ and TGFβ₃, in supporting hESCsundifferentiated culture, was examined. Initially, two measures wereused to estimate the ability of hESCs to grow in several culturesystems, namely percentage of differentiation and rate of growth. Theculture system used were: (1) feeder layer-free method based onfibronectin or Matrigel™ which are the most used matrices; (2) MEF, and(3) foreskin fibroblast. Based on these two parameters, the mediasupplemented with TGFβ₃, D2, HA16 and HA19, were found to be the mostsuitable to support undifferentiated hESC proliferation in all testedculture methods. Culture medium supplemented with 10 ng/ml bFGF only,failed to support hESC prolonged culture, in all the tested cultureconditions. Although 60% of the hESCs remained at the undifferentiatedstage in these conditions for a few passages, the proliferation rate waslow and with each passage the number of surviving hESCs decreased andthe percentage of background differentiation was increased.

D1 medium on a feeder layer-free system is capable of maintaining allhESCs features along with high proliferation rate—When cultured in thefeeder layer-free systems in the presence of the D1 medium, which issupplemented with TGFβ₁, the hESCs maintained all hESCs featuresincluding high proliferation rates. When cultured on the tested feederlayers in the presence of the D1 medium, the hESCs demonstrated arelatively high background differentiation rate of 20% and lowproliferation abilities as compared to hESCs cultured at the same feederlayers systems with the D2 HA19 or HA16 medium.

D1, D2 and HA16 media in feeder layer-free are capable of maintaininghESCs in a proliferative, undifferentiated state, with chromosomalstability and pluripotency—Human ESCs grown in the presence of the D1,D2 or HA16 medium in feeder-layer free conditions were culturedcontinuously for up to 53, 24 or 10 passages, respectively, whilemaintaining their ESC features, including undifferentiatedproliferation, chromosomal stability (as test by karyotype analysis, notshown) and pluripotency. The background differentiation rates were foundto be less than 10%, which is similar to the differentiation ratesoccurring when hESCs are cultured in the traditional culture systembased on MEFs as the feeder layer and medium supplemented with serumreplacement and 4 ng/ml bFGF [Amit et al, 2000]. Examples ofundifferentiated colonies cultured with D1, D2 or HA16 medium infeeder-layer free conditions and with the D2 or HA16 medium with thetested feeder layers are illustrated in FIGS. 1a -d.

hESCs cultured on feeder layer-free systems in the presence of the D1 orthe D2 medium are devoid of autofeeder—Interestingly, when the hESCswere cultured in either the D1 or D2 medium on the feeder layer-freesystem the cells did not differentiate at the periphery of the coloniesand did not form an outgrowth of feeder-like cells (also called“autofeeder”) (FIG. 1d ), as described in other reports on feederlayer-free culture methods for hESCs (Xu et al, 2001). No morphologicaldifferences could be observed between colonies grown in the feederlayer-free culture system and those grown with feeder layers (FIGS. 1a-d). Correspondingly, morphological features remained unchanged on asingle-cell level, rendering cells small and round, and exhibiting highnucleus-to-cytoplasm ratio, with a notable presence of one to threenucleoli and typical spacing between the cells (FIGS. 1a-d ).

The D1, D2 or HA16 media are capable of maintaining hESCs with normalpopulation doubling—Similar to cells grown on MEFs, cells cultured withD2 or HA16 medium in all tested culture methods, and the D1 medium inthe feeder layer-free systems, were passaged routinely every four to sixdays, at the same ratio of 1/2 or 1/3, indicating a similar populationdoubling time as of hESCs grown on MEFs. The cells were passage at thesame seeding efficiency of about 1 million cells per 10 cm², with thesame viability rate of over 95%. Using 40% human serum and 10% DMSO,cells were successfully frozen and thawed.

Karyotype analysis revealed normal karyotype of hESCs grown with the D1,D2, CM100 or HA16 media—15 passages and more after transferring thecells into the tested environments, karyotype analysis was performed byGiemsa banding on two separate cultures, representing the four mediumconditions, D1, D2, CM100 and HA16 at the different culture methods. Atleast 20 cells were tested from each sample, 40 cells from each mediumcombination. All examined cells were found to sustain normal karyotypeof 46,XX for cell lines I3 and I4 and 46,XY for cell line I6 (data notshown). Overall, these results suggest that the cells' karyotype remainsstable in the tested conditions, similarly to ESCs grown with MEFs usingtraditional methods (Amit et al, 2000).

hESCs cultured with the D1, D2 or HA16 express typical cell surfacemarkers—Several surface markers typical of primate undifferentiated EScells were examined using immunofluorescent staining (Thomson et al,1995, 1996, 1998). hESCs cultured with the D1, D2 or HA16 medium formore than 20 passages, while using the tested culture conditions, werefound to be strongly positive to surface markers TRA-1-60 (FIG. 2a ),SSEA4 (FIG. 2b ), TRA-1-81 (FIG. 2c ) and Oct 4 (data not shown). As inother primate ES cells, staining with SSEA3 was weak and negative forSSEA1 (data not shown).

hESCs cultured with the D1, D2 or HA16 medium are pluripotent as testedby EBs formation in vitro—The developmental potential of the cells afterprolonged culture in the tested culture methods was examined in vitro bythe formation of embryoid bodies (EBs). After more than 15, 20 and 30passages in medium D1, D2 and HA16, respectively, hESCs formed EBssimilar to those created by hESCs grown on MEFs (not shown). Withinthese EBs, stem cells differentiated into cell types representative ofthe three embryonic germ layers as described for EBs formed from hESCscultured on other culture systems (Itskovitz-Eldor et al, 2000).

EBs formed from the hESCs cultured on the D1, D2 or HA16 medium arecapable of differentiating into the ectoderm, endoderm and mesoderm celllineages—While undifferentiated cells cultured in the tested medium,feeder layers and matrices, expressed undifferentiated genetic markerssuch as Oct 4, Nanog, Sox2, Rex1, Cx43 and FGF4 (not shown)[Bhattacharya et al, 2004], cells harvested from 10 day-old EBsexpressed genes such as albumin and glucagon (endoderm), α-cardiacactin, β-globulin and Flk1 (mesoderm), and AC133 and neurofilament(ectoderm) as demonstrated by RT-PCR analysis (data not shown).

hESCs cultured with the D1, D2 or HA16 medium are pluripotent as testedby teratomas formation in vivo—The cells pluripotency was also tested invivo by teratomas formation. hESCs cultured for over 12 passages in theHA16, D1 or D2 medium, in the tested culture systems formed teratomasfollowing their injection into SCID-Beige mice. Within these teratomas,hESCs differentiated to representative tissues of the three embryonicgerm layers including; cartilage, muscle, bone and fat (mesoderm),stratified epithelium, melanin containing epithelium (ectoderm), andkidney like structure (endoderm and mesoderm), and epithelium ofendoderm origin (data not shown). Teratomas formation rates of 90%, andthe number of injected cells were identical to those demonstrated bycells cultured using traditional methods (Amit et al, 2000).

The HA16 and D2 media are suitable for derivation of hESC line onforeskin fibroblast feeder layers in a complete xeno-free system—Themedium combinations of the present invention were also tested for theability to support hESC line derivation. Using the HA16 or D2 medium onforeskin fibroblasts as a supportive layer, new hESC lines weresuccessfully derived and maintained for at least 2 passages (in thepresence of the D2 medium) or at least 18 passages (in the presence ofthe HA16 medium). The hESC line derived on foreskin in the presence ofthe HA16 culture medium demonstrated stem cells morphology at passage 18(and the culture is still ongoing), normal XY karyotype and pluripotencyas evidenced by the formation of EBs (FIGS. 3a-b and data not shown).The growth and success rates were similar to those obtained while usingtraditional culture methods. Since the used foreskin fibroblasts line,F21, were derived without any animal products, this new hESC lines werederived under complete xeno-free conditions. Thus, the new hESC linesexhibit typical hESC morphology and proliferation rates, normalkaryotype and pluripotency as evidenced by the formation of EBs.

Altogether, these results demonstrate that hESCs cells subjected toprolonged culture in the tested culture systems demonstrated all hESCsfeatures including; pluripotency, chromosomal stability, expression ofspecific genes and surface markers and indefinite proliferation asundifferentiated cells.

Example 2 The TGF β-Containing or IL6RIL6-Containing Culture Medium areCapable of Supporting Culturing of hESCs in Cell Suspension

Since the new TGFβ-containing culture medium which is described inExample 1, hereinabove, is designed for massive cell culture (lowprotein content), suitable for industrial and clinical proposes cellproduction, the ability of D1, D2, HA19 and HA16 media to supportsuspension culture of undifferentiated hESCs was examined. In addition,the ability of a medium containing the IL6RIL6 chimera, such as theCM100F or HACM100 medium (as described in the General Materials andExperimental Methods) to support a suspension culture of hESCs was alsoexamined. It should be noted that while the CM100F medium contains serumreplacement, the HACM100 medium is serum or serum replacement-free andthus presents a well-defined culture, xeno-free culture medium.

Experimental Results

The CM100F, HA16, D1, D2 and HA19 media are suitable for culturing hESCsin suspension—hESCs were cultured in suspension using the newlydeveloped TGFβ-containing medium types which are devoid of serum, serumreplacement and albumin. To date, the highest passage of hESCs grown insuspension in the tested medium types were 3 passages in the D1 medium,7 passages in the D2 medium, 10 passages in the HA19 medium and 17passages in the CM100F medium. All hESCs exhibited undifferentiatedmorphology at these passages and can be further cultured in these mediaand maintain hESCs features. Histological sections of the hESCs clumpsformed in the suspension cultures illustrated homogeneous cellpopulation, of round cells with large nucleus (FIGS. 5a-g ). Inaddition, when the cells were plated back on MEFs, they created colonieswith typical hESCs morphology (FIGS. 5b-e ), and if returned tosuspension cultures, they continued proliferation as undifferentiatedcells (data not shown). When hESCs were cultured in a suspension culturein the presence of the serum or serum replacement-free,IL6RIL6-containing HACM100 medium, the cells were expanded andmaintained in the undifferentiated state for at least 1-2 passages (datanot shown).

hESCs cultured in suspension in the presence of the D1, D2, HA19 orCM100F media express markers of undifferentiated hESCs—Cells cultured insuspension in the presence of the D2 medium for 3 passages as smallclumps of 200-1500 cells expressed stem cells markers such as Oct 4(FIG. 4a ), TRA-1-60 (FIG. 4b ), TRA-1-81 (FIG. 4c ) and SSEA4 (data notshown). Similar results were obtained with the CM100F, D1 or D2 mediumat passage 5 (p-5) (data not shown). When cultured in suspension culturein the presence of the CM100F or the HA19 medium the cells expressedhigh levels of typical stem cells markers such as Oct 4 (FIG. 6a ), Rex1(FIG. 6b ), Sox2 (FIG. 6c ), Nanog (FIG. 6d ) and FGF4 (data not shown)as demonstrated by RT-PCR analysis.

ESCs cultured in suspension are capable of forming EBs—When removed fromthe D1, D2 or HA16 medium and transferred to EBs medium (80% DMEM/F12supplemented with 20% FBSd and 1 mM L-glutamine), the cells formed EBscontaining representative tissues of three embryonic germ layers (asevidenced by histological analysis, data not shown).

Rhesus ESCs can be also cultured in the suspension cultures of thepresent invention—Similar results with Rhesus ESCs (monkey embryonicstem cells, line R366.4, University of Wisconsin, primate center,Thomson lab, Madison, Wis.), which are regarded as good candidate fortransgenic model to human diseases, were obtained when the Rhesus ESCswere cultured in suspension in the HA16, D1 and D2 TGFβ-containingculture media (data not shown).

Thus the new TGFβ-containing medium, which is devoid of serum, serumreplacement and albumin, or the IL6RIL6-containing medium are capable ofsupporting the undifferentiated culture of hESCs, while maintaininghESCs characteristics, and provide methods for massive culture of thesecells for industrial and clinical purposes.

Analysis and Discussion

hESCs, like mouse ES cells, are traditionally cultured with MEFs, whichmay expose them to animal pathogens. In this study, the presentinventors have demonstrated, for the first time, a defined animal, serumand feeder layer-free culture system for hESCs, based on the use ofcommercial medium supplemented with either TGFβ₃ or TGFβ₁ and bFGF, andhuman fibronectin matrix as substitute. This medium is designed formassive cultivation of cells in GMP for industrial or clinical purposes.All medium types of the present invention (with TGFβ₃ or TGFβ1) supporthESCs culture. When using the culture medium with TGFβ isoform 3 theresults are better; less background differentiation. All media types ofthe present invention support the culture with feeders as good as withthe regular serum containing media. Cells retained the sameproliferation rates and the same background differentiation percentagesas hESCs cultured with MEFs using traditional culture methods.Furthermore, the medium can also be used for massive suspended cultureof undifferentiated hESCs.

Two isoforms of TGFβ, TGFβ₃ and TGFβ₁, were tested for their ability tomaintain hESCs in an undifferentiated state using various cultureconditions. TGFβ₃ (D2 and HA16 media) was found to be the most suitablemedium supplement, supporting undifferentiated culture of hESCs whileusing all the tested culture possibilities. All hESCs, from threedifferent cell lines, continued to proliferate while retaining normalhESC features throughout the prolonged culture. Medium supplemented withTGFβ₁ (D1 medium) on the contrary, was demonstrated to supportundifferentiated hESC culture only while using feeder layer free culturesystems.

Cells cultured while using these media (D1, D2, and HA16) maintained allthe characteristics of ESCs. After prolonged culture of more than 20passages, the cells remained undifferentiated, as demonstrated by thecolony and single cell morphology, and by the expression of markerstypical of undifferentiated primate ESCs [Thomson et al, 1995, 1996,1998; Reubinoff et al, 2000]. In addition, while cultured in theseconditions, hESCs expressed specific markers for the undifferentiatedstage such as Oct 4, Sox 2, Rex1 and Nanog, as demonstrated by RT-PCR.

Karyotype analysis carried out on representative cell samplesdemonstrated that the hESCs' karyotype remained stable in the proposedconditions. None of the examined cells exhibited any karyotypeabnormalities.

The cells' pluripotency was examined in vitro. Cells cultured in thetested culture systems for more than 10 passages, formed EBs similar tothose created when grown on MEFs [Itskovitz-Eldor et al, 2000]. RT-PCRanalysis demonstrated that cells within these EBs differentiated intodifferent cell types representative of the three germ layers.Furthermore, following their injection to SCID-Beige mice, hESCscultured in the presence of the D1 and D2 media formed teratomascontaining a multitude of tissues types. hESCs cultured in the presenceof the HA16 medium also formed teratomas and their histologicalevaluation is in process. The teratoma formation rates were identical tothose of cells cultured with MEFs. Thus the pluripotency of the cellsculture continuously in the tested culture methods remained intact.

Additionally, and most importantly, the same measurements were used tocharacterize cells cultured with the D1, D2 and HA16 media insuspension. Cell culture under these conditions for more than 7passages, exhibit undifferentiated markers and when transferred todifferentiation promoting conditions, demonstrated pluripotency. Thusthese media can enable massive culture of undifferentiated hESCs, andfacilitate to development of control bioprocesses in industrialbioreactors.

These results demonstrate that hESCs can be maintained asundifferentiated cells in the proposed defined animal- and serum-freemedium combination, without any feeder cells (D1, D2 and HA16) oralternatively, with commonly used acceptable feeder layers (D2 andHA16). Thus, these media can facilitate hESCs culture for research,industrial and clinical purposes. Moreover, this novel culture media wasfound to support suspended culture of undifferentiated hESCs, the firstand primary step in developing a massive culture system for their growthand scale-up, a crucial step for any industrial and clinical uses.

The mechanism by which hESCs self-maintain is still unclear.Accumulating data suggest the involvement of TGFβ family members inhESCs renewal [Amit et al, 2004; Ludwig et al, 2006; James et al, 2005;Chen et al, 2006, Valdimarsdottir & Mummery, 2006]. Furthercomplementary research is required to explain the underlying mechanismsof action of TGFβ at the level of signal transduction, and the fact thatTGFβ₃ is more potent than TGFβ₁.

Future clinical uses of hESCs will require a reproducible, well-definedand xeno-free culture system. The culture method described in this studywhich uses fibronectin as a feeder-free matrix and D1, D2 or HA16 mediumand foreskins fibroblast meet these needs. The well-defined mediademonstrated in the present study are suitable for culturing hESCs andmay be advantageous for undertaking research on the mechanisms of ESCself-maintenance, especially of the possible roles of the TGFβ pathway.Other studies using hESCs, such as the research on differentiationpathways and mechanisms, will benefit from the availability of awell-defined and reproducible culture system.

Thus, the present invention discloses for the first time:

1. A culture system that allows hESC culturing in suspension asundifferentiated without carrier.

2. A scalable culture system, suitable for developing controlbioprocesses in industrial bioreactors.

3. A xeno-free system suitable for both culture and derivation of hESCs.Derivation of new hESC lines directly in suspension.

4. Three defined medium combinations, highly effective in supportinghESCs culture in variety of culture conditions. Priority of TGFβ₃ overTGFβ₁. TGFβ₃ was never demonstrated to promote self-renewal of stemcells.

Example 3 Prolonged Culturing of Pluripotent, Undifferentiated Human ESCells in Suspension in the Presence of the IL6RIL6 Chimera

Materials and Experimental Methods

hESC Cultures

hESC lines I-3, I-4 and I-6 [Amit & Itskovitz-Eldor, 2002] were culturedwith inactivated MEF for 54-89 passages as previously described [Amit etal, 2000]. The following culture medium combinations were tested fortheir ability to support the suspended culture of hESCs:

Basic culture medium—consisting of 85% DMEM/F12 (Biological Industries,Biet Haemek, Israel), containing 15% knockout serum replacement (SR), 2mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acidstock, and 4 ng/ml bFGF (all but mentioned are Invitrogen Corporationproducts, Grand Island N.Y., USA). This basic culture medium was usedfor the routine culture of hESCs in 2D culture with MEFs as control.

CM100F medium—consisting of the basic culture medium supplemented with100 ng/ml IL6RIL6 chimera [Chebath J, et al., 1997 and WO 99/02552 toRevel M., et al. SEQ ID NO:31].

CM6* medium—consisting of the basic culture medium with 100 ng/ml IL6and 0.5 ng/ml IL6 soluble receptor (both from R&D Systems MinneapolisMinn., USA).

CMLIF medium—consisting of the basic culture medium supplemented with1000 units/ml human recombinant leukemia inhibitory factor (LIF) (R&DSystems Minneapolis Minn., USA).

Culture in non-dynamic (static) suspension culture—The hESCs wereremoved from their culture dish using 1.5 mg/ml type IV collagenase(Worthington biochemical corporation, Lakewood, N.J., USA), furtherbroken into small clumps using 200 μl Gilson pipette tips, and culturedin suspension in 58 mm Petri dishes (Greiner, Frickenhausen, Germany) ina cell density of 1×10⁶—5×10⁶ cells/plate. The medium in the suspensionculture was changed daily, and the cells were passaged every 5-7 dayseither by manual cutting using 27 g needles or by gentle pippeting using20 μl Gilson pipette tips.

Culture in Erlenmeyer (dynamic suspension culture)—Cells cultured insuspension for at least one passage were transferred to 125 mlErlenmeyer (Corning Incorporated, Corning N.Y., USA) in 25 ml CM100Fmedium, and shaked continuously at 90 rpm using shaker (S3.02.10L, ELMIltd, Riga, Latvia). Medium was changed daily. Every 5-7 days the clumpswere broken with gentle pippetation and split in a ratio of 1:2.

Immunohistochemistry—Undifferentiated hESCs grown in suspension orre-cultured on MEFs and differentiated cells dissociated usingtrypsin-EDTA from 10-day-old EBs were fixed with 4% paraformaldehyde andexposed to the primary antibodies overnight at 4° C. Cys 3 conjugatedantibodies (Chemicon International, Temecula Calif., USA) were used assecondary antibodies (1:200). The primary antibodies (1:50) includestage-specific embryonic antigen (SSEA) 1, 3 and 4 (Hybridoma Bank,Iowa, USA), tumor recognition antigen (TRA) 1-60 and TRA1-81 (ChemiconInternational, Temecula Calif., USA), Oct4 (Santa Cruz Biotechnology,Santa Cruz, Calif., USA), β-tubulin (Chemicon International, Temecula,Calif., USA), troponin (Chemicon International, Temecula, Calif., USA),PSA-NCAM (Chemicon International, Temecula, Calif., USA).

Karyotype analysis—Karyotype analysis (G-banding) was performed on atleast 10 cells from each sample, two samples per test, as previouslydescribed [Amit et al, 2003]. Karyotypes were analyzed and reportedaccording to the “International System for Human CytogeneticNomenclature” (ISCN).

EB formation—For the formation of EBs, hESCs were passaged as describedand transferred to 58 mm Petri dishes (Greiner, Frickenhausen, Germany).EBs were grown in medium consisting of 80% DMEM/F12 (BiologicalIndustries, Biet Haemek, Israel), supplemented with 10% FBSd (HyClone,Utah, USA), 10% serum replacement (SR), 2 mM L-glutamine, 0.1 mMβ-mercaptoethanol, and 1% non-essential amino acid stock (InvitrogenCorporation, Grand Island N.Y., USA). 10-14 day-old EBs were harvestedfor RNA isolation and histological examination. For staining they wereplated on gelatin and cultured for 7-14 additional days. Forhistological analysis EBs were fixed in 10% neutral-buffered formalin,dehydrated in graduated alcohol (70%-100%) and embedded in paraffin. 1-5□μm sections were deparafined and stained with hematoxylin/eosin (H&E).

RT PCR—Total RNA was isolated from hESCs grown for 10, 15 and 20passages in suspension and from 10-14 day-old EBs formed from cellsgrown in suspension or cells cultured on MEFs) using Tri-Reagent (Sigma,St. Louis Mo., USA), according to the manufacturer's instructions. cDNAwas synthesized from 1 μg total RNA using MMLV reverse transcriptaseRNase H minus (Promega, Madison Wis., USA). PCR reaction includeddenaturation for 5 minutes at 94° C. followed by repeated cycles of 94°C. for 30 seconds, annealing temperature (as shown in Table 1) for 30seconds and extension at 72° C. for 30 seconds. PCR primers and reactionconditions were as described in Table 1 hereinabove, except that 30cycles of amplifications were performed for the Nanog, Rex1, FGF4 andSox2 PCR products. The PCR products were size-fractionated using 2%agarose gel electrophoresis. DNA markers were used to confirm the sizeof the resultant fragments.

Real time PCR—RNA was isolated from undifferentiated cells cultured onMEFs and from cells cultured in suspension for 10, 15 and 20 passagescontinuously. First-strand cDNA were synthesized as described above(RT-PCR). TaqMan Universal PCR Master Mix and Assay-on-Demand AgeneExpression Probes (Applied Biosystems, Foster City, Calif., USA) forOct4 and β-actin were used according to the manufacturer's guidelines.The reaction was performed with Applied Biosystems 7000 DNA SequenceDetection System (Applied Biosystems, Foster City, Calif., USA)according to the manufacturer's instructions. The relative expression ofOct4 was normalized to the expression of β-actin for the same sample.The cDNA of cells cultured on MEFs were used as calibrators, and therelative expression of Oct4 was calculated accordingly by using thestandard curve method described by the manufacturer. Three biologicalrepeats were conducted for each sample.

Flow cytometry—The clumps of hESCs cultured in suspension weredissociated to single cells using triple-Express (Invitrogen Corporationproducts, Grand Island, N.Y., USA). The cells were stained withanti-h/mSSEA4 Ab conjugated to Phycoerythrin, Phycoerythrin conjugatedRat IgG2B were used as isotype control [both from R&D systems,Minneapolis, Minn., USA]. The stained cells were then analyzed withFACScalibur flow cytometer (Becton Dickinson, San Jose, Calif., USA)using CellQuest software according to the manufacturer's instructions.

Teratoma formation—Cells from four to six 58 mm dishes were harvestedand injected into the hindlimb muscles of four week-old male of severecombined immunodeficiency (SCID)-beige mice. Ten weeks after theinjection the resultant teratomas were harvested and prepared forhistological analysis using the same method mentioned for EBs.

Western blot analysis—The cell pellets were lysed using RIPA buffer(Roche diagnostics, Penzberg, Germany) supplemented with phosphataseinhibitor cocktail (Sigma, St. Louis, Mo., USA). Proteins were extractedfrom I3 ESCs cultured in suspension for 29 passages, from cells culturedon MEFs only, from cells that were cultured in suspension for 10passages and then re-cultured on MEFs, and from cells of the triggergroup experiment. Total protein was measured by Bradford Protein Assay(Bio-Rad laboratories, Hercules, Calif., USA) according to themanufacturer's instructions. For Western blot analysis, the proteinswere separated on 6-10% gradient sodium dodecyl sulfate(SDS)-polyacrylamide (SDS-page) mini gel electrophoresis (Sigma, St.Louis, Mo., USA), and transferred to nitrocellulose membrane (Schleicher& Schuell, Dassel, Germany). After 1.5 hours blocking in 5% dry nonfatmilk (Nestle carnation, Switzerland), the membrane was incubated withprimary antibody for 2 hours at room temperature for β-actin (Sigma, St.Louis, Mo., USA), and overnight at 4° C. for gp-130, p-STAT-3, andSTAT-3 (All from Sigma, St. Louis, Mo., USA). The membrane was washedthrice with Tween-TBS (T-TBS) for 10 minutes, then incubated for 1 hourwith peroxidase-conjugated secondary antibody (Jackson Immuno research,Baltimore, Pa., USA), followed by incubation for 3 minutes withchemiluminescent substrate HRP (Pierce, Rockford, Ill., USA). Detectionwas performed using ECL western blotting analysis (Amersham PharmaciaBiotech, Piscataway, N.J., USA), and visualized by ImageMaster VDS-CL(Amersham Pharmacia Biotech, Bucks, England).

Trigger experiments—Forty-eight hours post splitting, cells cultured insuspension were transferred into the culture medium (control; basicculture medium) without the addition of the IL6RIL6 chimera. 24 hourslater, the chimera was added (at a concentration of 100 ng/ml) to theculture medium and cells were harvested immediately, after 30 minutes,after 3 hours and after 24 hours. Cells that were continuously culturedwith the IL6RIL6 chimera were used as control.

Apoptosis analysis—Apoptosis levels were examined by In Situ Cell DeathDetection Kit, AP (Roche Diagnostics GmbH, Mannheim, Germany) on the2^(nd), 4^(th), 6^(th), 8^(th), 10^(th) and 14^(th) days of continuousgrowth without splitting. Apoptotic cells were detected by incubationfor 3 minutes with Trypsin-EDTA (10×) (Invitrogen Corporation, GrandIsland N.Y., USA) of 1 ml cells per pellet, treatment with 4 ml Hank'ssolution and fixation on the dry slide of the single cells before usingthe kit. Apoptotic cells were counted by inverted Zeiss Axiovert 200fluorescent microscopy. At the same time, cell samples were harvestedfrom the same culture, broken with trypsin using the same method, andstained with trypan-blue to evaluate the number of viable cells. Cellswere counted by inverted Zeiss Axiovert 200 microscope.

Blocking of pg130 receptor experiments—Cells were cultured in suspensionwith CM100F medium. Increasing concentrations of anti-gp130 antibodies(Santa Crus Biotechnology, Santa Cruz, Calif., USA) of 100 ng/ml, 250ng/ml and 500 ng/ml, were added directly into the dishes on a dailybasis. The number of differentiated clumps was counted 6 days laterbased on morphology using IX70 Olympus inverted microscope.Characteristics of differentiated clumps included cyst formation, and aball like structures instead of disc like structures.

Experimental Results

The IL6RIL6 chimera, but not low concentrations of the IL6, soluble IL6or LIF, are capable of maintaining hESCs in an undifferentiated statewhen cultured on Fibronectin (2-D)—Three key cytokines of the IL6 family(LIF, IL6 and IL6RIL6 chimera) were tested for their ability to supportthe culture of undifferentiated hESCs in suspension. The ability of thevarious cytokines to maintain hESC self-renewal was first evaluated inthe 2-D feeder layer-free culture system using fibronectin as a matrix.While the background differentiation of hESCs cultured in the presenceof LIF or IL6 at the used concentrations (i.e., 1000 u/ml for LIF; 100ng/ml for IL6 and 0.5 ng/ml for soluble IL6 receptor) was as high as50%, it was only 22% on 2-D culture in the presence of the IL6RIL6chimera (at concentration of 100 ng/ml IL6RIL6 chimera), mainly bycreating “auto-feeders” as reported for other hESC feeder layer-freeculture systems (data not shown, demonstrated by FACS analysis). Using100 ng/ml of IL6RIL6 chimera and fibronectin as matrix it was possibleto culture the hESCs continuously for over 40 passages, whilemaintaining stable karyotype, expression of undifferentiation markersand EB and teratoma formation. An example of the cells cultured onfibronectin in the presence of the CM100F medium is illustrated in FIG.7 a.

The IL6RIL6 chimera, but not low concentrations of the IL6 and solubleIL6, are capable of maintaining cultures of hESCs in suspension in anundifferentiated state—The cytokines' ability to support the culture ofundifferentiated hESCs in suspension was then examined. With mediumsupplementation of 100 ng/ml IL6 and 0.5 ng/ml IL6 soluble receptor(medium CM6*) the cells formed either EBs or neurosphere-like structureswithin five to seven days of culture (data not shown). Theneurosphere-like structures demonstrated round morphology when culturedin suspension, and several days after being plated on fibronectinneuron-like structures positively stained with either nestin orβ-tubulin were observed (data not shown). With 100 ng/ml of IL6IL6chimera (the CM100F medium), the cells created disc-like structures 24hours after being cultured in suspension (FIG. 7c ). Histologicalsections of these clumps revealed a homogenous population of small cellswith large nuclei (FIG. 7d ). The clumps were split every 5-7 days whilemaintaining their morphology for at least 54 passages (i.e., for atleast a year of continuous culture). The three cell lines utilized forthis experiment, I-3, I-4 and I-6 hESCs showed no morphologicalvariability. When re-cultured on MEFs or fibronectin after 10 or 25passages in suspension, 100% of the clumps adhered to the MEFs or thefibronectin and after 24-48 hours demonstrated typical hESC colonymorphology, exhibiting high nucleus-to-cytoplasm ratio with a notablepresence of one to three nucleoli and with typical spacing between thecells (FIGS. 7e and f ). Differentiation background of the hESCscultured in suspension in the presence of the CM100F medium consisted ofup to 5% and included neurosphere-like structures as shown in FIG. 7 b.

hESCs cultured in suspension in the presence of the IL6RIL6 chimeraexhibit normal karyotype—Karyotype analyses by Giemsa banding wascarried out on each cell line after 23, 18 and 36 passages insuspension, for I-6, I-3 and I-4, respectively, and were found to benormal (data not shown). Similar results were obtained when the cellswere re-cultured on MEFs; all samples but one of I-3 (passage 12 on MEFsafter 10 passages in suspension) demonstrated normal karyotype. Theoriginal culture of I-3 that remained in suspension demonstrated normalkaryotype. Thus the karyotype of the suspended cell culture remainedstable.

hESCs cultured in suspension in the presence of the IL6RIL6 chimeraexhibit expression pattern of undifferentiated state—Several surfacemarkers typical of primate undifferentiated ESCs were examined usingimmunofluorescence staining [as described in Thomson et al, 1995, 1996,1998]. Human ESCs cultured in suspension with CM100F medium for 42 and43 passages were found to be strongly positive to surface markers SSEA4,TRA-1-60 and TRA-1-81 and Oct 4 (FIGS. 8a-d ). As in other primate ESCs,staining with SSEA3 was weak and negative for SSEA1. Similar resultswere obtained for the cells cultured in suspension and returned to theMEFs. The former were further tested for typical undifferentiationmarkers using RT-PCR. Similarly to cells cultured with MEFs, cellscultured in suspension for 10, 15 and 20 passages expressed geneticmarkers of pluripotency Oct 4, Nanog, Sox2, Rex1, and FGF4 (FIG. 9)[Bhattacharya et al, 2004]. No difference was detected between cellscultured for various durations, nor between cells re-cultured on MEFsafter continuous culture in suspension.

Flow cytometry analysis for SSEA4 revealed that 94.5% of I-4 hESCs, atpassage 30 in suspension (in the presence of the CM100F medium) and94.7% of I-6 hESCs at passage 20 in suspension (in the presence of theCM100F medium) were positive for SSEA4. For I-3 hESCs, 87.8% of thecells at passage 34 in suspension (in the presence of the CM100F medium)were positive for SSEA4, demonstrating a slight increase in backgrounddifferentiation (FIGS. 10a-c ). When the background differentiationincreased, differentiating clumps were removed using dissectingmicroscope based on their morphology, and a high expression level (>90%,e.g., >95%) of undifferentiation markers such as SSEA4, was restored(data not shown). Real time PCR analysis for the Oct4 gene expressionlevel demonstrated no significant difference between cells cultured insuspension and those cultured on MEFs, and between different passages(10, 15, 20) of culture in suspension (FIGS. 11a-b ).

hESCs cultured in suspension in the presence of the ILRIL6 chimerapreserve their pluripotency as demonstrated by EBs' formation—Thedevelopmental potential of the cells after prolonged culture insuspension was examined in vitro by the formation of EBs. When cellscultured in suspension for over 20 passages were transferred toserum-containing medium where the Il6IL6 receptor chimera was removed,after a lag of 7-10 days, hESCs formed cystic EBs similar to thosecreated by hESCs grown with MEFs (FIGS. 12a-b ). Within these EBs, stemcells differentiated into cell types representative of the threeembryonic germ layers [Itskovitz-Eldor et al, 2000]. Cells harvestedfrom 21 day-old EBs expressed genes such as albumin (endoderm),α-cardiac actin, β-globulin and Flk1 (mesoderm), and AC133 andneurofilament (ectoderm) as demonstrated by RT-PCR (data not shown).

hESCs cultured in suspension in the presence of the ILRIL6 chimerapreserve their pluripotency as demonstrated by teratoma formation—Cellpluripotency in vivo was demonstrated by teratoma formation. Cellscultured in the presence of the CM100F medium in suspension for 9, 10,14 or 26 passages were injected into SCID Beige mice, representing thethree tested cell lines, and after 10 weeks tumors formed in all fourinjected mice. Within these teratomas tissues representative of thethree germ layers were observed (FIGS. 12a-b ). Similar results wereobtained when cells cultured for at least 10 passages in suspension werereturned to the MEFs and cultured for additional 5-10 passages (data notshown).

Culture kinetics was tested by measuring the clumps' average size everysecond day during continuous culturing of 14 days. On day 7 the diameterincreased from 150 μM to 300 μM, and on day 14 it was measured 500 μM(FIGS. 13a-d ). Each of these clumps contained 2×10⁵ live cells on day2; 3.32×10⁵ cells on the day of splitting (day 6); and 9×10⁵ cells onday 14. Mechanical passaging resulted in an apoptosis level of 16% (FIG.13e ). During the next six days the average level of apoptosis droppedto 4.8% and increased again to 30% on day 14 of continuous culture (FIG.13e ). As expected, most of the apoptotic and necrotic cells werelocated at the center of the clump due to limited diffusion (FIGS. 13f-h). The cells' viability was found to be 90% until day 10, and 80% on day14. The increase in the clumps' diameter and the low level of apoptosisindicate that days 5-7 are indeed the optimal time for splitting, assplitting of the cells at this time point prevents a great loss of cellsfor apoptosis and enables continuous cell proliferation.

Cells were cultured in suspension in the shaking Erlenmeyer for threemonths. An examination after one month showed that morphologically theclumps of disc-like, sphere structures remained similar to that of thecells cultured statically (FIG. 14a ), although their size seemed morehomogenous. The average sphere diameter in the dynamic system was112±14.47 μM, each sphere containing 3.75×10⁴±3×10³ cells. Whenre-cultured on MEFs the clumps re-attached, formed typical colonies ofhESCs as occurred with cells that are cultured statically using Petridishes (FIG. 14b ), and were positively stained with undifferentiationmarkers (FIGS. 14c-e ). Culturing of the spheres in Petri dishes withmedium supplemented with serum without the Il6IL6 receptor chimeraresulted in EB formation, and when the latter were re-plated on gelatinthe cells were positively stained for troponin, PSA-NCAM, insulin andβ-tubulin (FIGS. 14f-h ). The karyotype of I3 hESCs cultured for onemonth in the Erlenmeyer was found to be normal. Finally, the cells'proliferation was tested to evaluate the suitability of this culturesystem for mass production of hESCs. The total sphere number increasedfrom 1.33×10⁴±461 on the seeding day to 3.5×10⁵±2.8×10⁴ after 10-11 daysin the dynamic culture, a 25-fold increase, and the total cell numberincreased from 5×10⁸ to 1.31×10¹⁰.

In order to gain insight into the possible contribution of the IL6RIL6chimera to the self-maintenance mechanism of hESCs, trigger experimentswere conducted. A clear increase in phosphorylated STAT3 levels could benoted three hours after re-adding the IL6RIL6 chimera, followed by anincrease in gp130 receptor levels, as demonstrated by Western blotanalysis (FIGS. 15a-d ). The increase in phosphorylated STAT3 and gp-130receptor levels could still be noted after 24 hours. Additional supportfor the IL6RIL6 chimera's involvement in the cells' self-maintenance wasobtained from a competition experiment where anti-pg130 antibody wasadded to the culture medium with increasing concentrations. The level ofbackground differentiation increased in parallel to the increasedantibody concentration from 5% when no antibody was added, to 67% when500 ng/ml antibody was added (FIG. 16). FIGS. 17a-b depict examples ofdifferentiated (FIG. 17b ) and undifferentiated (FIG. 17a ) hESC clumpsin the presence of the anti-gp 130 antibody.

Example 4 Testing Additional Media for Culturing Human ESCs inSuspension

Materials and Experimental Methods

Culturing Media for Human ESCs in Suspension Cultures

yFIL25+—85% DMEM/F12 (Biological Industries, Biet Haemek, Israel),containing 15% knockout serum replacement (SR), 2 mM L-glutamine, 0.1 mMβ-mercaptoethanol, 1% non-essential amino acid stock, 4 ng/ml bFGF (allbut mentioned are Invitrogen Corporation products, Grand Island N.Y.,USA), 25 ng/ml IL6 and 25 ng/ml sIL6-R (R&D systems, Minneapolis, Minn.,USA).

yFL3—85% DMEM/F12 (Biological Industries, Biet Haemek, Israel),containing 15% knockout serum replacement (SR), 2 mM L-glutamine, 0.1 mMβ-mercaptoethanol, 1% non-essential amino acid stock, 4 ng/ml bFGF (allbut mentioned are Invitrogen Corporation products, Grand Island N.Y.,USA), and 3000 u/ml human recombinant leukemia inhibitory factor (hrLIF)(R&D Systems Minneapolis Minn., USA).

yFL2—85% DMEM/F12 (Biological Industries, Biet Haemek, Israel),containing 15% knockout serum replacement (SR), 2 mM L-glutamine, 0.1 mMβ-mercaptoethanol, 1% non-essential amino acid stock, 4 ng/ml bFGF (allbut mentioned are Invitrogen Corporation products, Grand Island N.Y.,USA), and 2000 u/ml hrLIF (R&D Systems Minneapolis Minn., USA).

yFL1—85% DMEM/F12 (Biological Industries, Biet Haemek, Israel),containing 15% knockout serum replacement (SR), 2 mM L-glutamine, 0.1 mMβ-mercaptoethanol, 1% non-essential amino acid stock, 4 ng/ml bFGF (allbut mentioned are Invitrogen Corporation products, Grand Island N.Y.,USA), and 1000 u/ml hrLIF (R&D Systems Minneapolis Minn., USA).

TLF—85% DMEM/F12 (Biological Industries, Biet Haemek, Israel),containing 15% knockout serum replacement (SR), 2 mM L-glutamine, 0.1 mMβ-mercaptoethanol, 1% non-essential amino acid stock, 4 ng/ml bFGF (allbut mentioned are Invitrogen Corporation products, Grand Island N.Y.,USA), 1000 u/ml hrLIF (R&D Systems Minneapolis Minn., USA) and 0.12ng/ml TGFβ₁ (R&D Systems Minneapolis Minn., USA).

Experimental Results

New media combinations are suitable for culturing human ESCs insuspension culture—Four additional culture medium were tested for theability to support suspension culture of hESCs; TLF, yFIL25+, yFL3 andyFL1. Using these culture media the human ESCs created spheres, ordisc-like structures, 24 hours after being cultured in suspension usingnon-dynamic culture conditions (FIG. 18a-e ). The clumps were splitevery 5-7 days while maintaining their morphology, and as of the day ofwriting this report reached 31, 19, 18 and 18 passages for TLF, yFIL25+,yFL3 and yFL1 respectively. Two lines of hESCs (I3 and I4) were testedwith each of the TLF, yFIL25+, yFL3 media, except for the yFL1 mediumwhich was tested only with the I3 hESC line. When hESCs which werecultured for 10 passages in suspension with TLF or yFIL25+ media werere-cultured on MEFs or fibronectin, 100% of the clumps adhered to theMEFs or fibronectin and after 24-48 hours demonstrated typical hESCcolony morphology, exhibiting high nucleus-to-cytoplasm ratio with anotable presence of one to three nucleoli and with typical spacingbetween the cells (FIG. 18c ).

Human ESCs cultured in suspension in the presence of the TLF, yFIL25+,or yFL3 culture media exhibit normal karyotype—Karyotype analyses byGiemsa banding were carried out on each cell line after 18 passages inthe TLF medium, 14 passages in the yFIL25+ medium and 14 passages in theyFL3 medium and were found to be normal.

Human ESCs cultured in suspension in the presence of the TLF, yFIL25+,yFL3 or yFL1 media exhibited undifferentiated state as evidenced by theexpression of the surface markers SSEA4, TRA-1-60 and TRA-1-81 and Oct4—Several surface markers typical of primate undifferentiated ESCs wereexamined using immunofluorescence staining according to the methodsdescribed elsewhere [Thomson et al, 1995, 1996, 1998]. Human ESCscultured in suspension with TLF, yFIL25+, yFL3 and yFL1 medium for 31,18, 18 and 18 passages, respectively, were found to be strongly positiveto surface markers SSEA4, TRA-1-60 and TRA-1-81 and Oct 4 (FIGS. 19a-d).

Human ESCs cultured in suspension in the presence of the TLF, yFIL25+,yFL3 or yFL1 culture media express markers of pluripotency—Human ESCscultured in suspension under non-dynamic culture conditions were furthertested for typical undifferentiation markers using RT-PCR analysis.Similarly to cells cultured with MEFs, cells cultured in suspension for18, 16, 16 or 18 passages in the presence of medium TLF, yFIL25+, yFL3or yFL1, respectively, expressed genetic markers of pluripotency Oct 4,Nanog, Sox2, Rex1, and FGF4 (Data not shown).

Human ESCs cultured in suspension in the presence of the TLF, yFIL25+,yFL3 or yFL1 are capable of forming ebs which represent all threeembryonic germ layers—The developmental potential of the cells afterprolonged culture in suspension was examined in vitro by the formationof EBs. When cells cultured in suspension for over 10 passages weretransferred to serum-containing medium where the growth factors wereremoved, after a lag of 7-10 days, hESCs formed cystic EBs (Data notshown). Within these EBs, stem cells differentiated into cell typesrepresentative of the three embryonic germ layers. Cells harvested from21 day-old EBs expressed genes such as albumin (endoderm), α-cardiacactin, β-globulin and Flk1 (mesoderm), and AC133 and neurofilament(ectoderm) as demonstrated by RT-PCR (Data not shown).

Analysis and Discussion

Undifferentiated hESCs are traditionally cultured in 2D on eitherfeeder-layer cells or on acellular matrix. This study demonstrates forthe first time a method of culturing these cells as free floatingspheres for prolonged periods in medium consisting of serum replacement,bFGF, and IL6RIL6 chimera.

Mouse ESCs can be cultured continuously without feeder layers providedthat the culture medium is supplemented with leukemia inhibitory factor(LIF), which was found to be involved in the self-maintenance of mouseESCs [Smith et al, 1988; Williams et al, 1988; Rose et al, 1994; Conoveret al, 1993; Niwa et al, 1998]. Accumulating data regarding hESCssuggest that LIF has no effect on preventing hESC differentiation[Thomson et al, 1998; Reubinof et al, 2000]. Furthermore, activation ofkey proteins of the LIF cellular pathway, such as STAT3 was found to beweak or absent in hESCs [Daheron et al, 2004; Sato et al, 2004]. Anadditional candidate for hESC self-maintenance is the IL6RIL6 chimera.While LIF acts through the heteromeric complex of LIF-receptor andgp130, the IL6RIL6 chimera requires only gp130 to activate theintracellular pathway involving activation of JAK kinases and STATtranscription factors. In mouse cells, the IL6RIL6 chimera is known tobe a potent inducer of the LIF/IL6 pathway, which results in a higherresponse compared with the effect caused by IL6 alone or even thechimera components added separately (IL6 and IL6 soluble receptors IL6R)(Chebath et al, 1997).

Furthermore, to date the only method for deriving new mouse ESC lines infeeder layer-free conditions is based on the addition of factors fromthe IL6 family [Nichols et al, 1994], or the combination of LIF and BMP4when serum-free conditions are used [Ying Q L et al, 2003]. Of the IL6family, the IL6RIL6 chimera was demonstrated as the most potent factorin supporting the feeder layer-free isolation of mouse ESC lines[Nichols et al, 1994]. The chimera has a much higher affinity for humangp130 than the mixture of IL6 and sIL6R [Kollet et al, 1999].Nevertheless, a recent study demonstrated that although it activates theLIF/STAT pathway in hESCs, on its own the IL6RIL6 chimera isinsufficient to maintain hESC pluripotency in adhesive two-dimensional(2D) feeder layer-free culture [Humphrey et al, 2004].

In the present study several cytokines of the IL-6 family (IL6RIL6chimera, LIF and IL6) were tested for their ability to maintain hESCs intheir undifferentiated state. Medium supplemented with 100 ng/ml of theIL6RIL6 chimera when used in conjunction with 4 ng/ml bFGF supported theculture of three hESC lines in suspension for over 40 passages andretained normal hESC features, including the expression of surfacemarkers and genes typical of undifferentiated hESCs as detected by FACS,RT-PCR and immunostaining, normal karyotype and teratoma formation.

Similar results were obtained when the cells were transferred back ontothe MEFs, including one group which was transferred to and from the MEFsand the suspension for four times and remained stable throughout.

The mechanism by which hESCs self-maintain is not entirely understood.In mouse ESCs the role of LIF and other members of the IL6 family,acting through gp130 and the JAK/STAT3 pathway, in maintaining prolongedculture of undifferentiated ESCs is well known [Smith et al, 1988;Williams et al, 1988; Rose et al, 1994; Conover et al, 1993; Niwa et al,1998]. Previous studies did not demonstrate a significant effect of theIL6 family, including a fusion protein of portions of IL6 and the IL6receptor, on the self-maintenance of undifferentiated hESCs [Daheron etal, 2004; Humphrey et al, 2004; Sato et al, 2004]. In this study thepresent inventors demonstrate that the IL6RIL6 chimera does support theculture of hESCs in suspension and to a lesser extent in adhesionculture with fibronectin serving as matrix. Trigger experimentsdemonstrate that IL6RIL6 chimera indeed increases the STAT3phosphorylation levels in both suspension and 2D cultures (data notshown). Blocking the IL6RIL6 chimera effect by anti-gp130 antibody,increased the level of differentiation, further indicating that theIL6RIL6 chimera is involved in the self maintenance of the cells.

Further research is required to both elucidate the underlying mechanismsof action of the IL6RIL6 chimera at the level of signal transduction,its time course and intensity at which different pathways (JAK/STAT,PI-3 kinase, MAPK, see Hirano et al, 1997) are activated, and tounderstand why this pathway is less effective when undifferentiatedcells are cultured in 2D.

The described culture system was also found to support hESCsproliferation. The size of each clump during passaging (5-7 days) wasfound to increase by 1.5 folds, and associated with low apoptosis levelsand high viability rates. The number of cells in each sphere increased1.66-fold during each passage (5-7 days). Taking together, the kineticfeatures of the newly developed culture system indicate that the systemis as proficient as the 2D culture systems and could be used as a basefor routine culture of undifferentiated hESCs.

An important requirement for clinical and industrial application ofhESCs is a scalable culture system capable of generating masses ofcells. The culture system presented here, can also be used as a base forthe mass production of undifferentiated hESCs using dynamic systems suchas spinner flasks and bioreactors.

Shifting undifferentiated hESCs from adhesion to suspension willfacilitate the development of controlled scale-up processes. Inaddition, the methodology presented here requires Petri dishes and doesnot require enzymatic splitting, supportive cells nor conditioned media,making it a cost-effective system. Coupled with its simplicity, thisapproach is an attractive option for the routine culture of hESCs.

The present inventors' experience with the Erlenmeyer's dynamic systemindicate that hESCs can be cultured continuously and maintain theirtypical features, while enabling a scale-up of 25 folds in 10 days.Although seeding concentrations and medium metabolites should still beoptimized, the results presented here demonstrate that this system couldserve as a basis for developing a controlled process for mass productionof hESCs in bioreactors.

To date, only one publication reports a successful culture of mouse ESCsin a suspension system for one passage, which resulted in a 31 foldexpansion in 5 days [Cormier J T., et al, 2006; Tissue Eng. 2006November; 12(10:3233-45]. The system was based on medium supplementedwith calf serum and 1000 u/ml LIF. A more recent publication by the samegroup demonstrates that the same culture system could also be used forsomewhat prolonged culture by splitting the cells with trypsin [ZurNieden et al, 2007]. However, under these conditions, the mESCsexhibited a doubling time of 15 hours which may lead to chromosomalinstability. Nevertheless, the authors did not show karyotype analysisof the cultured ESCs. As shown in several publications, mouse and humanESCs share only some of their features; they differ in the inability toculture hESCs in 2D using the traditional medium supplemented with calfserum and LIF without a feeder layer [Thomson et al, 1998]. The abilityof the IL6RIL6 chimera to support the self-maintenance of hESCs insuspension illuminates once again the question of LIF-STAT3 pathway'spossible role in hESC self-renewal.

Thus, this culture system presented herein is a further step forwardtoward creating the culture conditions that will make possible tofulfill the promise of hESCs.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

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What is claimed is:
 1. A method of expanding and maintaining humanpluripotent stem cells in an undifferentiated state, the methodcomprising culturing the human pluripotent stem cells in a feedercell-free culture, comprising a defined culture medium which comprisesan IL6RIL6 chimera and basic fibroblast growth factor (bFGF) for atleast one passage, wherein said medium is devoid of serum, wherein saidIL6RIL6 chimera comprises the amino acid sequence set forth by SEQ IDNO: 33, said culture medium is capable of maintaining the humanpluripotent stem cells in an undifferentiated state, and wherein saidhuman pluripotent stem cells are capable of differentiating into theendoderm, mesoderm and ectoderm embryonic germ layers, thereby expandingand maintaining human pluripotent stem cells in the undifferentiatedstate.
 2. The method of claim 1, wherein said culturing is for at least15 passages.
 3. The method of claim 1, wherein said culture medium isserum placement-free.
 4. The method of claim 1, wherein said culturemedium comprises serum replacement.
 5. The method of claim 1, whereinsaid culture medium is devoid of animal contaminant.
 6. The method ofclaim 1, wherein said culturing is effected in suspension.
 7. The methodof claim 6, wherein said suspension is devoid of substrate adherence. 8.The method of claim 1, wherein said culturing is effected on afeeder-layer free matrix.
 9. The method of claim 8, wherein saidfeeder-layer free matrix is a fibronectin matrix.
 10. The method ofclaim 1, wherein said IL6RIL6 chimera is provided at a concentration ofat least 25 ng/ml.
 11. The method of claim 1, wherein said bFGF isprovided at a concentration of at least 2 ng/ml.
 12. The method of claim1, wherein said at least one passage occurs after 4-6 days of culturingin said culture medium.
 13. A method of deriving a human embryonic stemcell line, the method comprising: (a) obtaining a human embryonic stemcell from a pre-implantation stage blastocyst, post-implantation stageblastocyst and/or a genital tissue of a fetus; and (b) culturing saidhuman embryonic stem cell in a feeder cell-free culture, comprising adefined culture medium which comprises an IL6RIL6 chimera and basicfibroblast growth factor (bFGF) for at least one passage, wherein saidmedium is devoid of serum, and wherein said IL6RIL6 chimera comprisesthe amino acid sequence set forth by SEQ ID NO: 33, said culture mediumis capable of maintaining the human embryonic stem cells in anundifferentiated state, thereby deriving the embryonic stem cell line.14. A method of generating lineage-specific cells from pluripotent stemcells, the method comprising: (a) culturing the pluripotent stem cellsin a feeder cell-free culture, comprising a defined culture medium whichcomprises an IL6RIL6 chimera and basic fibroblast growth factor (bFGF)for at least one passage, wherein said medium is devoid of serum, andsaid IL6RIL6 chimera comprises the amino acid sequence set forth by SEQID NO: 33, said culture medium is capable of maintaining the pluripotentstem cells in an undifferentiated state, wherein said pluripotent stemcells are capable of differentiating into the endoderm mesoderm andectoderm embryonic germ layers, to thereby obtain expanded,undifferentiated pluripotent stein cells; and (b) subjecting saidexpanded, undifferentiated pluripotent stem cells to culturingconditions suitable for differentiating and/or expanding lineagespecific cells; thereby generating the lineage-specific cells from thepluripotent stem cells.
 15. The method of claim 14, further comprisingisolating lineage specific cells following step (b).
 16. A method ofgenerating embryoid bodies from pluripotent stem cells, the methodcomprising: (a) culturing the pluripotent stem cells in a feedercell-free culture comprising a defined culture medium which comprises anIL6RIL6 chimera and basic fibroblast growth factor (bFGF) for at leastone passage, wherein said medium is devoid of serum, said IL6RIL6chimera comprises the amino acid sequence set forth by SEQ ID NO: 33,said culture medium is capable of maintaining the pluripotent stem cellsin an undifferentiated state, and wherein said pluripotent stem cellsare capable of differentiating into the endoderm, mesoderm and ectodermembryonic germ layers, to thereby obtain expanded, undifferentiatedpluripotent stem cells; and (b) subjecting said expanded,undifferentiated pluripotent stem cells to culturing conditions suitablefor differentiating said embryonic stem cells to embryoid bodies:thereby generating the embryoid bodies from the pluripotent stem cells.17. The method of claim 16, wherein said pluripotent stem cells arehuman pluripotent stem cells.
 18. A method of generatinglineage-specific cells from pluripotent stem cells, the methodcomprising: (a) culturing the pluripotent stem cells in a feedercell-free culture, comprising a defined culture medium which comprisesan IL6RIL6 chimera and basic fibroblast growth factor (bFGF) for atleast one passage, wherein said medium is devoid of serum, said IL6RIL6chimera comprises the amino acid sequence set forth by SEQ ID NO: 33,said culture medium is capable of maintaining the pluripotent stem cellsin an undifferentiated state, and wherein said pluripotent stem cellsare capable of differentiating into the endoderm, mesoderm and ectodermembryonic germ layers, to thereby obtain expanded, undifferentiatedpluripotent stem cells; (b) subjecting said expanded, undifferentiatedpluripotent stem cells to culturing conditions suitable fordifferentiating said expanded, undifferentiated pluripotent stein cellsto embryoid bodies; and (c) subjecting cells of said embryoid bodies toculturing conditions suitable for differentiating and/or expandinglineage specific cells; thereby generating the lineage-specific cellsfrom the pluripotent stem cells.
 19. The method of claim 18, whereinsaid pluripotent stem cells are human pluripotent stem cells.
 20. Afeeder cell-free cell culture comprising a human pluripotent stem celland a defined culture medium, said culture medium comprising an IL6RIL6chimera and basic fibroblast growth factor (bFGF), wherein said mediumis devoid of serum, wherein said IL6RIL6 chimera comprises the aminoacid sequence set forth by SEQ ID NO: 33, wherein said culture medium iscapable of maintaining said human pluripotent stem cell in anundifferentiated state for at least one passage, and wherein said humanpluripotent stem cells are capable of differentiating into the endoderm,mesoderm and ectoderm embryonic germ layers.